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The list of abbreviations
Abbreviations
Full name
ALS
Acid-labile subunit
AKT
Protein kinase B
ADT
Androgen deprivation therapy
AI
Androgen independent
APES
3-aminopropyl triethoxy-saline
AR
Androgen receptor
BAD
BCL-2 associated death promoter
BCL-2
B-cell CLL/lymphoma 2
bFGF
basic fibroblast growth factor
BLAST
Basic local alignment search tool
BPH
Benign prostatic hyperplasia
BSA
Bovine serum albumin
CAM
Chorioallantoic membrane
cDNA
Complementary DNA
C-FABP
Cutanious fatty acid binding protein
CK
Cytokeratin
DAB
3, 3’-Diaminobenzidine
DBD
DNA binding domain
DEPC
Diethylpyrocarbonate
DHT
5-α-dihydrotestosterone
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Abbreviations
Full name
DMSO
Dimethyl sulphoxide
DNA
Deoxyribonucleic acid
EBM
EndoGRO basal medium
ECL
Enhanced Chemiluminescence
ECM
Extra cellular matrix
EDTA
Ethylenediaminetetraacetic acid
EGF
Epidermal growth factor
EGFP
Enhanced green fluorescent protein
EHS
Engelbreth-Holm-Swarm
ET-1
Endothelin-1
EZH2
Enhancer of zeste homologue 2
FABP
Fatty acid binding protein
FGF
Fibroblast growth factor
FISH
Fluorescence in-situ hybridization
Flt-1
Fms-like tyrosyl kinase-1
FCS
Foetal calf serum
G418
Geneticin
GSTP1
Glutathione S Tranferase π
Grb2
Growth factor receptor-binding protein 2
HPC
Hereditary prostate cancer gene
HIF
Hypoxia-induced factor
HRP
Horse radish peroxidise
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Abbreviations
Full name
HUVEC
Human umbilical vein cells
IFGBP
Insulin-like growth factor binding protein
IGF
Insulin-like growth factor
IRS1
Insulin receptor substrate-1
KDR/Flk-1
Kinase insert domain receptor
LB
Lysogeny broth
LBD
Ligand binding domain
LCFA
Long chain fatty acid
LOH
Loss of heterozygosity
LUTS
Lower urinary tract symptoms
MAPK
Mitogen-activated protein kinase
MVD
Microvessel density
NBF
Neutral buffered formalin
NCRI
National cancer research institute
NES
Nuclear export signal
NLS
Nuclear localization signal
OD
Optical density
P13K
Phosphorylates phosphatidylinositol 3’-kinase
PAP
prostatic acid phosphatase
PBS
Phosphate buffered saline
PCR
Polymerase chain reaction
PDB
Protein data bank
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Abbreviations
Full name
PDK
Phosphatidylinositol-dependent kinase
PDK1
3-phosphoinositide dependent protein kinase 1
PEN-STREP
Penicillin/streptomycin
PIN
Prostatic Intraepithelial Neoplasia
PIP3
Phosphatidylinositol (3,4,5)-trisphosphate
PlGF
Placental growth factor
PPARs
Peroxisome proliferator-activated receptors
PPRE
Peroxisome proliferator response element
PTEN
Phosphatase and tensin homologue
PTHrP
Parathyroid hormone related protein
PSA
Prostate specific antigen
RANK
Receptor activator of nuclear factor - 
RB
Retinoblastoma susceptibility
RIN
RNA integrity number
RNA
Ribonucleic acid
rpm
round per minute
RT
Room temperature
RT-PCR
Reverse transcription polymerase chain reaction
RXR
Retinoic acid receptor
SC
Subcutaneous
SD
Standard deviation
SHC
Src- and collagen-homology
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Abbreviations
Full name
siRNA
Short interfering RNA
SOB
Super optimal broth
SOC
SOB medium plus glucose
SV40
Simian virus 40
TBS
Tris-buffered salin
T/E
Trypsin/EDTA
TMB
3, 3’, 5, 5’-Tetramethylbenzidine
TSG
Tumor suppressor gene
uPA
urokinase-type plasminogen activator
VEGF
Vascular endothelial growth factor
min (s)
Minute (s)
g
Gram
mg
Milligram
µg
Microgram
ng
Nanogram
pg
Picogram
ml
Millilitre
µl
Microlitre
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Chapter 1 Introduction
1.1 The epidemiology of prostate cancer
1.1.1 Incidence and mortality of prostate cancer
Prostate cancer is the most common form of cancers in men in the UK. Nearly 35,000
men are diagnosed with prostate cancer in the UK each year and the figure is still going
up due to longer life expectancy of men. It accounts for approximately a quarter (24%)
of all new male cancer diagnoses.
Although there has been a pronounced rise in prostate cancer incidence over the last 20
years, the increase in mortality rate has been much more slowly. Much of the increase in
incidence was due to the increased detection of prostate cancer by a wider use of
prostate specific antigen (PSA) testing. Prostate cancer accounts for around 13% of
male deaths from cancer in the UK and is the second most common cause of cancer
mortality in men, after lung cancer. It has been reported 10,000 deaths in the UK from
prostate cancer in 2005 as shown in Figure 1.1.
Figure 1.1
Age standardised incidence and motility rates for prostate cancer 1975–2005 [1, 2]
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1.1.2 Worldwide Epidemiology
Geographic variations exist in the impact of prostate cancer. Clinical incidence and
mortality of prostate cancer are low in Asian men, higher in population of developed
western countries and highest in African-Americans [3-5]. Interestingly, once
individuals from a low incidence/mortality region move to a high incidence/mortality
region, the risk of prostate cancer becomes higher. The length of time living in the new
environment seems also to be correlated with the increase [6, 7].
1.1.3 Factors affecting the prostate cancer
1.1.3.1 Age
The prostate cancer has been known as a disease of elderly men. The diagnosis is very
rare before the age of 50 years. However, beyond this age both incidence and mortality
rate increased exponentially. The risk of prostate cancer increases faster with age than
any other major cancer and, with a growing age population grow due to longer life
expectancy, the burden of illness from prostate cancer will probably continue to
increase in the future. The studies have shown that the age specific prevalence of
histologic prostate cancer is similar in Japan and United States but there is a remarkable
difference in the age specific prevalence of clinical prostate cancer between Japanese
and American men [8]. These data indicated that the initiation rate of prostate cancer
may be the same in both groups however there are differences in the rate of promotion
or progression to clinically evident prostate cancer. Therefore, the presence of histologic
prostate cancer appears to be related to age, other risk factors that increase the
development of prostate cancer probably affect the promotion or progression steps of
the malignant transformation pathway.
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1.1.3.2 Genetic factors
There is an increase in prostate cancer incidence in male relatives of patients with
prostate cancer – Cater et al. showed that prostate cancer was diagnosed about two
times (15% of men who had a father or brother affected by the prostate cancer) higher
than in control group (8% of control population with no affected relatives) [9]. It has
also been reported that a significant number of early onset prostate cancers may be
inherited in an autosomal dominant fashion and suggest that hereditary factors are most
commonly affected men with early onset of prostate cancer, however, they are
responsible for only a small proportion of prostate cancer [9].
Genetic linkage analysis on potential genetic susceptibility regions which include the
hereditary prostate cancer gene 1 (HPC1) on 1q23-25, hereditary prostate cancer gene 2
(HPC2) on 17p, hereditary prostate cancer gene X (HPCX) on Xq27-28 and
Predisposing for Cancer Prostate (PCAP) on 1q42-43 have identified several strong
candidate genes such as RNASEL, ELAC2, and MSR1 [10-15]. A number of studies
provide support, both functional and epidemiological, that these genes play a role in
hereditary prostate cancer. Yet other studies have suggested that their role may be small.
Further work will be required in order to understand how these genes involved in
prostate cancer and to sort out the roles of the various variants of these genes [16].
1.1.3.3 Dietary factors
Dietary patterns have been recognised as one of the key factor in the development of
different type of cancer malignancies. Consumed food component may be metabolized
to oncogene, may alter hormonal balance, or may protect against the development of
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certain cancer. As mentioned previously (section 1.1.3), the rising rate of prostate
cancer in both black South African and Japanese men who migrated to the United States
is thought to be due to the change in their dietary patterns, and risk was higher when
they migrated at a younger age. As the “Westernization” diet pattern is closely linked
with high dietary intake of fat, the increased risk for the development of prostate cancer
has a relationship with dietary fat, and it has been proved by several studies that dietary
factors in black, white, and Asian-Americans with prostate cancer found a positive
association between prostate cancer risk and total fat intake in all ethnic groups [7].
Similarly, for mortality, a multination survey highly correlated prostate cancer deaths
with total fat consumption [17, 18] (see Figure 1.2). Some studies propose that obese
men have increased prostate cancer mortality by 2.5-fold [7].
Figure 1.2
The positive correlation between age-adjusted mortality for prostate
cancer and animal fat consumption by country [19].
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Further evidence suggested that not only the amount of fat consumption but also the
type of fat had an effect on prostate cancer growth. Dietary fats are classified as either
saturated fatty acids or unsaturated fat acids as can be seen in Table 1.1. It has been
suggested that saturate fat consumption has been constantly associated with prostate
cancer risk [20] whereas some studies have been published on the role of monounsaturated fats in association with protecting prostate cancer [21]. An epidemiological
research also noted that in countries where people consume larger amount of olive oil
which is rich in all kinds of mono-unsaturated fats, a lower incidence of several cancers
including prostate cancer was found [19]. In addition, a number of researches have been
performed on effect of various kinds of poly-unsaturated fatty acids. A negative effect
of fish oil, a highly unsaturated omega-3 fatty acid, on tumour development was
suggested when nude mice fed menhaden (omega-3 rich fish oil concentrate) had
decreased growth of injected Du145 prostate cancer cells [22].
Saturated Fatty Acids
CH3(CH2)16COOH
(No double bond)
Unsaturated Fatty Acids

Mono-unsaturated
CH3(CH2)7CH=CH(CH2)7COOH
(One double bond)

Poly-unsaturated
CH3(CH2)4CH=CHCH2CH=CH(CH2)7COOH
(Two or more double
CH3CH2CH=CHCH2CH=CHCH2CH=CH
bond)
(CH2)7COOH
Table 1.1
Classification of fatty acids
Page | 10
Retinoic acid, also known as vitamin A, is a lipophilic molecule that plays an important
role in regulating cell growth, differentiation, cell-cycle arrest and apoptosis [23-25].
Retinoic acid shows the capacity of reducing tumourigenicity and is currently used as a
therapeutic agent in several human cancers [26]. It has been also reported that intake of
retinoic acid is inversely proportional to prostate cancer incidence [27, 28]. However,
results of some studies were conflicting – in some tissues retinoic acid appears to
promote rather than inhibit cell survival and retinoic acid treatment can enhance skin
tumour formation [29-31]. Recent studies from Schug et al. demonstrated that retinoic
acid mediated cell biological process by two contradictory signalling pathways: retinoic
acid inhibits cell proliferation by signalling through its receptors; the ‘non-traditional’
pro-proliferative activities of retinoic acid may be mediated by nuclear receptor
PPARβ/δ. The response through witch pathway might depend on the ratio of
FABP5 / CRABP-II [32].
The relationship between other diet factors and prostate cancer is also complex. A
cohort study found that increasing consumption of beans, lentils, tomatoes, dates and
dried fruit significantly reduced risk for prostate cancer [33]. Studies also showed that
diets riching in soya protein had a protective effect on prostate cancer [34, 35]. In
general, the results from dietary intake studies support the concept that a high fibre and
low fat diet may protect men against the development of prostate cancer.
1.1.3.4 Other factors
The association of prostate cancer with other risk factors have been evaluated in a
number of studies. However, the statistic data for risk factors such as socioeconomic
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status, occupation, cigarette smoking, alcohol consumption and sexual activity were not
convincing that these risk factors resulted in an increasing incidence of prostate cancer.
1.2 The pathology of prostate cancer
1.2.1 Anatomy and physiology of the prostate gland
The prostate is an accessory gland and present only in males. The prostate is located in
front of the rectum, just below the urinary bladder and it surrounds the urethra, which
carries urine from the bladder out through the penis. Between the rectum and the
bladder, attached to the prostate are a pair of glands called the seminal vesicles which
provide nutrients for the semen (see Figure 1.3).
Figure 1.3
Interior of male urinary system and prostate (Picture is taken from
http://www.pioa.org/c_anatomy.html)
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Prostate is one of the exocrine gland which secrete through ducts to the outside of the
body and partly made up of muscular fibres and partly glandular, with ducts opening
into the prostatic portion of the urethra. The prostate gland secretes a slightly alkaline
fluid that contains a number of enzymes, citric acid, prostate specific antigen and zinc
[36-38]. The fluid neutralizes and protects sperm from the acidic environment of the
female vagina.
The prostate is a small acorn shaped gland and composes of four morphological regions
known as the anterior fibromuscular stroma, peripheral zone, central zone and transition
zone [39] (see Figure 1.4). The anterior fibromuscular stroma forms entire surface of the
prostate as the prostatic capsule and consists of approximately 5% of the prostatic
weight. This zone is usually devoid of glandular components because this zone is
composed only of muscular and fibrous tissue. The anterior fibromuscular stroma is
mainly responsible for the secretion of prostatic fluid during ejaculation by initiating
muscular contractions. The peripheral zone constitutes approximately 70% of the
normal prostate gland. It is the posterior portion of the prostate gland which surrounds
the distal urethra. More than 64% prostate cancer originated from this zone. The central
zone makes up 25% of the total tissue mass of the prostate gland. This zone surrounds
the ejaculatory ducts and accounts for only up to 2.5% of prostate cancers although
these cancers tend to be more aggressive [40]. The remaining 5% of the prostate gland
is formed by the transitional zone which is the innermost part of the prostate gland and
surrounds the urethra where it passes through the prostate. The transitional zone is the
primary site of Benign Prostatic Hyperplasia (BPH).
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Figure 1.4
Schematic of prostate gland describes zonal anatomy. PZ = peripheral
zone, TZ = transition zone, CZ = central zone [41]
1.2.2 Normal prostatic cells
The prostatic epithelium mainly consists of three anatomically distinct cell populations:
Luminal cell, basal cell and neuroendocrine cells as shown in Figure 1.5. The secretory
luminal cells are the most predominant cells in the prostate and responsible for secretion
of prostatic proteins including prostate-specific antigen (PSA) and prostatic acid
phosphatase (PAP), two important clinical biomarkers for prostate cancer. These cells
express androgen receptor (AR) and require androgens for maintenance of their function
and viability [42]. Luminal cells can be characterised by their expression of the filament
high molecular weight proteins, cytokeratins (CK) 8 and CK18. Most of the prostate
cancers are adenocarcinomas (95%) which are considered to differentiate towards
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secretory cells as the majority of cancer cells express luminal cell specific markers such
as CK8, CK18 and PSA [43].
Figure 1.5
Schematic view of the cell types within human prostate epithelium.
Picture is taken from [44]
Basal cells are the second major epithelial cell type in prostate. These cells are located
between the luminal cells and the underlying basement membrane and are able to
express high molecular weight protein markers such as CK5 and CK14. Unlike luminal
cells, the basal cell population neither produce prostatic secretory proteins nor express
AR and they do not undergo apoptosis in response to androgen ablation [45, 46].
However, it has been reported that basal cells express factors that have a protective
function against DNA damage, such as anti-apoptotic gene Bcl-2 and the free-radical
scavenger Gst-π [39]. In addition, it is speculated that the basal cell population contains
a subset of cells acts as a stem cell reservoir in order to generate all three types of cells
[47].
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Finally, the third prostatic epithelial cell type is the neuroendocrine cells which can be
found in all area of prostate. The marker used to distinguish this cell population is
Chromogranin A. Neuroendocrine cells provide specialized neuropeptides or
neuroendocrine factors that support the vitality and proliferation of luminal epithelial
cells [39]. Some studies indicated a positive correlation between the level of
neuroendocrine
factors
and
the
more
advanced
stage
of
prostate
adenocarcinoma [48, 49].
1.2.3 Prostate cancer initiation
1.2.3.1 Benign Prostatic Hyperplasia (BPH)
BPH has been characterized as non-malignant overgrowth of prostate tissue. BPH
shares some similarities with prostate cancer: (1) Both of them are age related disease;
(2) Both of them require androgen stimulation. However, BPH is not a precursor of
prostate cancer. BPH develops in the transition zone, a ring of tissue around the urethra
and its growth is inward toward the prostate's core, constantly tightening around the
urethra and associated with lower urinary tract symptoms (LUTS). Most of prostate
carcinoma, on the other hand, begin in the outer peripheral zone of the prostate and
invade the surrounding tissues. Although the pathogenesis of BPH is still poorly
understood, some studies indicated that BPH is related to hormonal changes that occur
as men age. The testis produces the hormone testosterone, which is converted to
5-alpha-dihydrotestosterone (DHT) and estrogen in certain tissues. High level of DHT,
a testosterone derivative involved in prostate growth, may trigger for initiation of BPH
[50].
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1.2.3.2 Prostatic Intraepithelial Neoplasia (PIN)
PIN is the earliest morphologically identifiable stage in prostatic carcinogenesis and is
defined as abnormal proliferation of secretory cells of prostatic glands without invasion
into surrounding stroma [51]. PIN has been classified into two groups: low grade and
high grade PIN. In low grade PIN, cell nuclei are enlarged, remarkable vary in size,
have a normal or slightly increased chromatin content and possess small or
inconspicuous nucleoli [52]. In high grad PIN, cell nuclei are also enlarged but at the
similar size and shape variation, have dramatically increased density and clumping of
chromatin content and possess prominent nucleoli that are similar to those of invasive
carcinoma cells. In addition, the basal cell layer shows frequently interruptions in high
grade PIN but intact or rarely interrupted in low grade PIN.
Currently, conventional use of term PIN without qualification refers to high grade PIN
which must be included as a part of pathologic report. Four main patterns of high grade
PIN have been distinguished: flat, tufting, micropapillary and cribriform [53]. Tufting is
the most common pattern found in PIN lesion (97%) although multiple patterns can be
found in most of cases at the same time. There are some common properties between
high grade PIN and prostate cancer: epidemiologically, the incidence and extend of both
high grade PIN and prostate cancer is associated with age; histopathologically, high
grade PIN and prostate cancer are both multifocal and share a same location in
peripheral zone; Cytogenetic studies showed that they also share similar genetic
alterations and proliferation activity. Many studies exhibited correlation between PIN
and prostate cancer and the appearance of high grade PIN is an important marker for
detection of potential prostate cancer [54].
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1.2.4 Gleason grading system of prostate cancer
Gleason score, a grading system for prostate cancer devised by Dr. Donald Gleason in
1977 as a method for evaluating the prognosis of men with prostate cancer, is the most
widespread method of prostate cancer tissue grading used today [55]. The classic
Gleason scoring diagram identified five distinct prostate cancer patterns that are
technically referred to as tumour grades which have been numbered ranging from 1 to 5,
with Grade 1 being the least aggressive and Grade 5 being the most aggressive cancer
(see Figure 1.6).
Figure 1.6
Gleason
grading
system
diagram
(Picture
is
taken
from
http://en.wikipedia.org/wiki/Gleason_staging_system)
Gleason score is a sum of the primary grade representing the dominant of tumour
pattern and a secondary grade which is assigned to second most prevalent tumour
pattern and is a number ranging from 2 to 10. The Gleason grading system allows
patients with tumours of similar biologic behaviour to be compared in studies and as a
strong prognosis factor, it also provides useful information for patient treatment options.
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1.2.4 Prostate cancer cell lines
To understand the pathogenesis and malignant progression of prostate cancer, prostate
cancer cell lines are wildly used in the laboratory studies. However, the most common
used prostate cancer cell lines are: PNT2, LNCaP, Du145 and PC-3.
1.2.4.1 PNT2
The cell line is derived from a normal human prostate of a 33 year old male at post
mortem and has been immortalized by transfection with a plasmid containing simian
virus 40 (SV40) with a defective replication origin [56]. The PNT cells possess a well
differentiated morphology with the expression of cytokeratin 8, 18 and 19 which are the
markers of differentiated luminal cells of the glandular prostate. Cytokeratin 14, a
marker of epithelial basal cells, is not expressed. In addition, the cells are not
tumorigenic when injected in Nude mice [57].
1.2.4.2 LNCaP
The cell line was established in 1977 by J.S. Horoszewicz et al., from a needle
aspiration biopsy of the left supraclavicular lymph node of a 50 year old male patient
with confirmed diagnosis of metastatic prostate carcinoma. Prostatic specific markers,
such as androgen receptor (AR), prostate specific antigen (PSA) and prostatic acid
phosphatise (PAP), are presented in cultured cells [58, 59]. LNCaP cells are androgen
sensitive, do not produce a uniform monolayer, but grow in clusters and they are
responsive to 5-alpha-dihydrotestosterone [60]. LNCaP cells rarely form tumours in
mice by subcutaneous inoculation and no metastatic spread was noted in any of the
animals given injections [61].
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1.2.4.3 DU-145
The cell line is derived from prostate carcinoma that was metastatic to central nervous
system lesion of a 69 year old patient. Not only cytokeratin markers of luminal cells of
the glandular prostate but also MUC1 are found in DU-145 cells [62]. However,
expression of prostatic associated markers (AR, PAP and PSA) are not present in this
cell line [62]. Tumours were formed in 29 out of 32 of nude mice given subcutaneous
injection of DU-145 cells but no evidence of metastasis has been found [63].
1.2.4.4 PC-3 and PC-3M
The cell line was initiated from a bone marrow metastasis of a grade IV prostatic
adenocarcinoma from a 62 year old male patient [64]. The cultured cells show androgen
independent growth and produce both high incidence of tumorigenicity and metastasis
in nude mice [65]. The PC-3M human prostate cancer cell line was isolated from liver
metastasis produced in nude mice subsequent to intrasplenic injection of the parental
PC-3 cells. As a derivative of PC-3 cell line, PC-3M shares some similar properties with
PC-3 but more aggressive than the rest of prostate cancer cell lines including PC-3 [66].
1.3
Oncogenes and tumor suppressor genes in prostate cancer
The molecular biological events involved in initiation, promotion and progression of
prostate cancer are still unclear. Apparently, it is generally believed that prostate cancer
formation and metastasis are due to mutations of specific genes which play important
roles in regulating cell cycle control, proliferation, differentiation and apoptosis. The
genes have been divided into two main groups: oncogenes and tumour suppressor
genes.
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1.3.1 Oncogenes in prostate cancer
Oncogenes can be defined as genes are partly or solely responsible for tumourigenesis.
Oncogenes are generally mutated forms of certain normal cellular genes known as
proto-oncogenes which provide signals to regulate cell biological behaviour such as cell
growth, cell differentiation and apoptosis. There are a large number of oncogenes are
now recognized, but only a relatively small number have been associated with prostate
cancer.
1.3.1.1 C-MYC oncogene
The C-MYC oncogene is located at 8q24 and involves in regulation of cell proliferation,
cell differentiation and apoptosis [67]. This well known oncogene, characterized as a
transcription factor, increases its copy numbers by amplification or/and chromosomal
gain [68]. Both over expression and amplification of C-MYC have been detected in
prostate carcinomas. Fluorescence in-situ hybridization (FISH) with a specific probe for
C-MYC was performed in PIN, localized prostate cancer and its metastasis [69]. A
number of studies also showed that the level of C-MYC mRNA is enhanced in prostate
cancer compared to BPH or normal prostate tissue. These results indicated that the
amplification of C-MYC may be potential marker for prostate cancer progression.
However, the contribution of C-MYC to prostate cancer development and progression is
infrequent: 8% of primary prostate cancers and about 30% of metastatic lesions.
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1.3.1.2 C-ERB-B2 oncogene
The C-ERB-B2 oncogene also known as HER-2/NEU is located at chromosome 17q1121. It encodes the p185neu transmembrane glycoprotein which belongs to the epidermal
growth factor receptor family of tyrosine kinase [70]. It has been reported that over
expression of C-ERB-B2 may contribute to androgen independence in prostate cancer
[71]. It has been reported that C-ERB-B2 can activate the androgen receptor pathway in
absence of ligands such as steroid hormones. In addition, casodex, an androgen
antagonist, blocks the effects of growth factors but not C-ERB-B2 on androgen receptor
function [71]. This indicates that either C-ERB-B2 signalling cascade produces a
molecule that binds at a competitively higher affinity than casodex, or C-ERB-B2
activates androgen receptor function on fresh non-optimal androgen receptor ligand
binding domains rather than to enhance androgen receptor function on high-affinity
binding sites. Sadasivan et al. found that C-ERB-B2 expression correlated with
advanced stages of prostate cancer and higher Gleason scores [72]. However, reports on
frequencies of C-ERB-B2 over-expression in prostate cancer vary wildly.
1.3.1.3 BCL-2 oncogene
The B-cell CLL/lymphoma 2 (BCL-2) oncogene is located at chromosome 18q21.3 and
is an inner mitochondrial membrane protein that plays an important role in regulating
cell apoptosis. It has been reported that BCL-2 promotes tumorigenesis by inhibiting
programmed cell death instead of increasing cell division [73]. In the prostate gland,
BCL-2 normally expressed in basal epithelial cells which are not affected by androgen
deprivation but not in luminal cells. In prostate cancer cells, BCL-2 oncogene contribute
to accelerating progression of androgen dependent LNCaP cells to androgen
Page | 22
independence via inhibiting castration-induced apoptosis initiated by androgen
deprivation [74]. Similarly, McDonnell et al. (1992) also demonstrated that transfection
of BCL-2 into LNCaP cells enabled the survival of those cells during androgen
withdrawal. These results indicated that over expression of BCL-2 prevented apoptosis
of prostate cancer cells subjected to androgen withdrawal and related to progression to
androgen independence in prostate cancer cells.
1.3.1.4 EZH2 polycomb oncogene
The enhancer of zeste homologue 2 (EZH2) oncogene is located at chromosome 7q35
and is known as a transcription repressor. It has been reported that the expression of
both EZH2 mRNA and protein progressively increase from BPH to primary prostate
tumor, to metastatic tumors, indicating that elevation of EZH2 protein level may accrue
in advance of the development of metastasis [75]. Furthermore, over expression of
EZH2 protein in prostate cancer cells leads to transcriptional repression as a result,
increases cell proliferation [76]. These results suggested that EZH2 protein level, as a
marker of prostate cancer progression and metastasis, might be helpful to predict patient
outcome after prostatectomy.
1.3.1.5 RAS oncogenes
The RAS oncogene family, consisting of Harvey (H) RAS (located at chromosome
11p15.5), Kirsten (K) RAS (located at chromosome 12p12.1) and neuroblastoma (N)
(located at chromosome 1p13.2), encodes cytoplasmic GTPase signal transduction
proteins with molecular weights of approximately 21kDa. The RAS proteins are
localized at the inner surface of the plasma membrane and are involved in regulating
Page | 23
cell proliferation, adhesion, apoptosis and cell migration activated by point mutations at
codon 12, 13 or 61 [77, 78]. Some studies indicated that over expression of RAS protein
seems to link to prostate cancer progression and could be correlated with development
of bone metastasis [79]. However, activation of RAS oncogenes is uncommon in
prostate cancer with a frequency of less than 5% mutant RAS oncogenes in North
American.
1.3.2 Tumor suppressor genes in prostate cancer
Tumor suppressor genes (TSGs) are normal genes involved in reducing cell
proliferation, promoting cell differentiation, repairing DNA mistakes and inducing cell
apoptosis. The importance of TSGs is that loss of function mutations of TSGs leads to
breakdown of cellular order and deregulation, which results in turning a normal cell into
a tumor cell. Mutations in TGSs are frequently seen in many types of cancers including
prostate cancer. There are three major ways to identify which TSGs are involved in
prostate cancer:
1. Identifying prostate cancer TSGs mutated in prostate cancer pedigrees and
positional cloning
2. Studying TSGs that had already been found to be important in other types of
cancers
3. Examining prostate cancer for regions of constantly chromosomal loss of
heterozygosity (LOH)
Similar to the study of oncogenes, the investigation of TSGs in prostate cancer will
provide insights into initiation and progression of prostate cancer. It will also bring new
prognostic markers and future treatment strategies.
Page | 24
1.3.2.1 P53 TSG
The p53 gene is located at chromosome 17q13 and encodes a 53kDa nuclear
phosphoprotein. The normal functions of p53 appear to be cellular growth suppression,
regulation of cell cycle which contributes to maintenance of DNA stability and cell
apoptosis [80, 81]. When the cell DNA damaged, p53 increasing the expression of
mitotic inhibitors such as p21/WAF1, which binds to and inhibits cyclin kinase
complexes, preventing phosphorylation of specific substrates and arresting the cell cycle
in G1 phases to allow for repairing damaged DNA. If the damaged DNA is
unrepairable, p53 induces apoptosis [82]. However, mutation of p53 TSG results in a
loss of p21 gene expression and loss of regulation of the cell cycle which enables
mutated cells to divide in an unchecked manner and increases tumourigenic potential.
The p53 TSG is inactivated by point mutations which are the most commonly observed
genetic alterations (50%) in human cancers analysed. In addition, the wild type p53
protein has a very short half-life within cells, however, the mutant p53 protein has a
prolonged half-life and it is detectable by immunohistochemistry.
In prostate cancer, the p53 protein has been widely examined using molecular and
immunohistochemical assay. No evidence of p53 activation has been found in BPH and
a low frequency (10-30%) of p53 TSG mutations has been observed in primary prostate
tumours. However, p53 activation rate ranges from 50 to 80% in patient with metastatic
prostate cancer [83, 84]. These results showed that mutations in the p53 TSG rarely
occur in early stage of prostate carcinoma and are more commonly activated in
advanced and metastatic prostate cancers. These evidences indicated that p53 TSG
activation is associated with malignant progression of prostate cancer and worse
prognosis.
Page | 25
1.3.2.2 RB TSG
The retinoblastoma susceptibility (RB) gene is located at chromosome 13q14 within one
of the most commonly deleted regions in prostate cancer. RB TSG encodes a nuclear
phosphoprotein which suppresses cell proliferation by arresting cell cycle in G1 phase
through its interactions with transcription factors, notably the E2F family. Loss of RB
occurred at relatively early stage of the tumour progression as the studies showed a
similar frequency in a low grade localised prostate cancer as well as in more advanced
prostate carcinoma [51]. Bookstein et al. (1990) showed that transfection of wild type
RB gene into a prostate cancer cell line Du145 resulted in a dramatic reduction (~15fold) in the tumor formation of the transfected cells in nude mice. The results above
suggested that RB TSG could play an important role in tumorigenicity in prostate
cancer. Furthermore, approximately one third LOH of the RB TSG were found in
prostate cancer cases. However, some data demonstrated the lack of correlation between
LOH at RB and absent RB protein expression, which indicated the existence of another
TSG in this region [85].
1.3.2.3 PTEN TSG
The phosphatase and tensin homologue (PTEN) TSG is located at chromosome 10q23.3
and
acts
as
a
(3,4,5)-trisphosphate
lipid
phosphatase
(PIP3)
by
that
inactives
dephosphorylation.
the
phosphatidylinositol
PIP3
initiates
the
phosphatidylinositol-dependent kinase (PDK) / AKT signaling pathway to increase cell
proliferation and cell survival [86]. In normal cells, PTEN expression blocks the
PDK/AKT signal transduction pathway that results in cell apoptosis. On the contrary, in
tumor cells, LOH and mutation of PTEN results in promoting AKT activity, thereby
blocking apoptotic signal.
Page | 26
It has been reported that PTEN LOH or/and mutation are rare in early stage of prostate
cancer whereas these changes are most commonly found in metastatic prostate cancer
[87]. McMenamin et al. also found that complete loss of PTEN is associated with highstage and grade prostate cancer. These results suggest that PTEN may play an important
part in the acquisition of metastatic potential in prostate cancer.
1.3.2.4 GSTP1 TSG
Glutathione S Tranferase π (GSTP1) TSG is located at chromosome 11q13 and encodes
the detoxifying isoenzyme GSTP1 which inactivates electrophilic carcinogens by
conjugation with glutathione. In addition, GSTP1 also protects DNA from oxidative
damage [88]. The studies demonstrated that the activity of GSPT1 in prostate tumors
was inhibited by hypermethylation which is the most common (>90%) reported
epigenetic alteration in prostate cancer [89]. It is also been reported that the
transcription of GSTP1 is completely blocked in the LNCaP cell line because of
hypermethylation of the gene promoter. Harden et al. demonstrated that the quantitation
of GSTP1 hypermethylation using real-time PCR is highly sensitive and specific to
prostate cancer (not present in BPH specimens or normal tissue), which results in
improving accuracy of the detection. As GSTP1 gene hypermethylation occurs in early
stage of prostate cancer, it may serve as a useful prognostic marker.
Page | 27
1.4
Molecular mechanisms of metastasis in prostate cancer
The main cause of prostate cancer treatment failure is metastasis which is defined as a
process that cancer cells escape from the site of primary tumour and form secondary
tumours at distant sites [90]. The molecular mechanism of metastasis in prostate cancer
is still not clear, however, there are a few hypotheses to explain the site specific pattern
of metastatic displacement. One of the most well known hypothesis is the “Seed and
Soil” theory [91] which consists of a number of steps:

Firstly, the malignant cancer cells lose their cell-cell and cell-matrix adhesive
characteristics, become motile and gain the capability to degrade proteins that
make up the surrounding extracellular matrix using degradative enzymes [92].

Secondly, once the malignant cancer cells escaped from the primary tumor and
enter the circulation (blood and/or lymphatic vessels), they interact with the host
immune system resulting cell apoptosis. However, very few cells survived in the
circulation known as the “seed”.

Finally, the surviving cancer cells arrest at the specific distant site known as the
“soil” because the environments of different organs are biologically unique. In
other words, their endothelial cells express different cell-surface receptors and
growth factors that may influence the cell-endothelial interactions. The cells
then extravasate from the circulation, invade to the interstitium and proliferate to
form a metastatic tumor. These processes require various changes in cell
properties, such as: cell proliferation, cell invasion, and angiogenesis [90].

In addition, the metastasis site also capable of producing further metastasis [93].
Page | 28
1.4.1 Mechanisms of bone metastasis in prostate cancer
The metastasis of prostate cancer shows a significantly strong predilection for bone. The
studies demonstrated that approximately 90% of advanced prostate cancer metastasizes
to bone [94]. The osseous tissue consists of two different structures: compact bone
which is mineralized cortical bone and trabecular bone which is metabolically active
cancellous bone. The outer layer of the trabecular bone contains bone marrow, where
the osteoblasts and osteoclasts are generated. The most frequent sites of skeletal
metastasis are the trabecular bones with high content of bone marrow [95].
1.4.1.1 Osteoclastic bone resorption
The first important steps of prostate cancer cells metastasis to bone is the activation of
osteoclasts resorption which results in not only allowing the seeding of cancer cells but
also releasing survival and growth factors that promote prostate cancer metastasis [96].
Among these factors, the most prominent is the Receptor Activator of Nuclear Factor  ligand (RANK ligand) produced by osteoblastic cells.
RANK ligand is mainly located on the surface of osteoblasts and bind to its specific
receptor (RANKR) which is expressed on the surface of the osteoclastic precursors. The
studies showed that osteotropic factors such as parathyroid hormone, 1, 25dihydroxyvitamin D3, and parathyroid hormone can induce osteoclast formation and
activation by up-regulation of RANKR [97]. Osteoprotegerin, a member of tumor
necrosis factor receptors, acts as a competitive inhibitor against RANK ligand binding
RANKR and thus blocks the RANKR - RANK ligand interaction [96]. On the other
hand, some contraventional results demonstrated that osteoprotegerin also bind to tumor
Page | 29
necrosis factor related apoptosis inducing ligand results in inhibition of cancer cell
apoptosis [98]. Nevertheless, osteoprotegerin remarkably decreases prostate cancer
metastasis to bones because it inhibits bone resorption [99]. Therefore, the development
of competitive antibodies, such as recombinant osteoprotegerin, against RANK ligand
can be potential treatments for bone metastasis (Figure 1.7).
Figure 1.7
The ratio of RANK ligand to osteoprotegerin determines the level of
osteoclastogenesis.
1.4.1.2 Domination of the osteoblastic lesions
Osteoblasts are bone forming cells which come from mesenchymal stem cells [100].
Many factors can regulate the growth, survival and differentiation of osteoblasts, such
as Endothelin-1 (ET-1), basic fibroblast growth factor (bFGF), Insulin-like growth
factor (IGF) and Parathyroid hormone related protein (PTHrP). Some of them are also
produced by metastatic prostate cancer cells.
Page | 30
ET-1, a peptide of 21 amino acid residues, is synthesized in vascular endothelial cells
and is involved in angiogenesis, the formation of the new bone and osteoblastic bone
metastasis [101]. The previous studies demonstrated that ET-1 is one of the most
overexpressed factors in osteoblast and can increase prostate cancer cell proliferation
probably by enhancing the mitogenic effects of IGF and epidermal growth factor.
Recent studies showed that overexpress ET-1 can up-regulate the vascular endothelial
growth factor (VEGF) expression through the endothelin receptor A in osteoblast and
thus could act as an angiogenic factor in the formation of bone metastasis [101].
IGF-1 plays an important role in cell proliferation and inhibiting cell apoptosis in
prostate cancer cells. Insulin-like growth factor binding protein-3 (IFGBP3) together
with acid-labile subunit (ALS) binds IGF-1(75% - 90% of circulating IGF-1) and the
ligands binding trigger the downstream pathway of IGF-1 receptor (IGF-1R). The
ligands binding activate the intrinsic tyrosine kinase of IGF-1R which results in
phosphorylation of IGF-1R substrates - insulin receptor substrate-1 (IRS1), Src- and
collagen-homology (SHC) and growth factor receptor-binding protein 2 (Grb2). Then,
these phosphorylated factors active with Ras – Raf – mitogen-activated protein kinase
(MAPK) cascade, which result in increasing cell proliferation [102]. The IRS-1 also
phosphorylates phosphatidylinositol 3’-kinase (P13K) and AKT, which promotes antiapoptotic survival of the cancer cells as shown in Figure 1.8. In addition, it has been
reported that elevated concentration of urokinase-type plasminogen activator (uPA)
which is produced by prostate cancer cells also stimulate the local activity of IGF-1,
therefore, increases prostate cancer cells proliferation and induces the osteoblastic
reaction.
Page | 31
Figure 1.8
IGF-I–IFGBP3–ALS binds to IGF-IR to trigger a series of ligandmediated activations including RAS–RAF–MAPK and PI3K–AKT
signalling pathway, controlling cell proliferation and cell apoptosis,
respectively. Picture is taken from [102].
1.5
Androgen
1.5.1 Hormone therapy for advanced prostate caner
Androgen, also known as androgenic hormones or testoids, has key influence on
regulating the development and maintenance male characteristics through binding to its
receptors. The predominant and most active androgen is testosterone which is mainly
(approximately 90%) synthesized by cells in testes and an important factor in the
development and function of the prostate gland.
Page | 32
In prostate cancer, radical prostatectomy is the most common treatment when the
disease is at localized stage. For patients diagnosed with advanced or metastatic disease,
androgen deprivation therapy (ADT) is the standard treatment which includes medical
or surgical castration, anti-androgens, and combined androgen blockade, thereby
resulting in a suppression of androgen signalling. ADT is an effective initial treatment
with 80%-90% primary response rate, however, the duration of response to ADT is
limited to 14 – 20 months (reviewed in [103]). Then, the prostate cancer become
refractory to additional treatment due to the fact that prostate cancer cells eventually
developed to an androgen independent (AI) state. In most cases with ADT, patients
developed castrate resistant prostate cancer, which is no longer in response to androgen
deprivation. Nevertheless, the majority of AI tumor cells remain expressing androgen
receptor (AR), which appears to act as normal in spite of the castrate level of androgen.
1.5.2 Multiple pathways to androgen independence
1.5.2.1 Androgen receptor
The testicular synthesized testosterone transports to prostate tissue and is converted into
5α-dihydrotestosterone (DHT), which has higher affinity for AR than testosterone, by a
5α-reductase enzyme. Altogether, testosterone and DHT bind to AR and exert their
biological function roles including regulation of specific gene transcription. The AR is a
member of the steroid hormone receptor family of ligand-dependent transcription factor.
Like other member of nuclear receptors, AR protein is composed of several functional
domains: (1) N-terminal regulatory domain, which mainly involved in most ligandactivated transcriptional activity; (2) DNA binding domain, which is the best conserved
region and important for recognizing androgen response elements; (3) hinge domain,
Page | 33
which connects the DNA binding domain and ligand binding domain and it also
contains a ligand dependent nuclear translocation signal; (4) ligand binding domain,
which is involved in androgens binding induced activity as well as in nuclear
localization, receptor dimerization and interaction with other proteins [104]. The
molecular mechanisms of prostate cancer progression from an androgen-dependent
phase to an androgen-independent stage are still not fully understood. However, there
are several signalling pathways may account for the development of androgenindependent prostate cancer such as: AR amplification, AR mutations and AR
coregulators overexpression.
1.5.2.2 Androgen receptor amplification
Amplification of the AR gene is a potent mechanism that enables prostate cancer cells
to become more sensitive to the reduced level of circulating androgens. It has been
reported that amplification of AR was rarely found in untreated primary prostate cancer.
However, approximately 30% of androgen-independent prostate tumors had increased
levels of AR gene expression after ADT (reviewed in [105]). Furthermore, studies have
found that increased level of AR gene expression in progression from androgen
sensitive to androgen resistance using xenograft models [106]. However, AR
amplification only occurred in a proportion of androgen independence prostate cancers
suggested that AR amplification is not the only mechanism responsible for development
of androgen independence.
Page | 34
1.5.2.3 Androgen receptor mutations
Studies have found that the frequency of mutations in the AR is significantly increased
in tumors collected after ADT compared with few AR mutations in primary tumor
samples without the therapy [107, 108]. These results suggested that gaining of AR
mutation may be one of the mechanisms leading to the development of prostate cancer’s
androgen resistance. AR may acquire transcriptional activity not only by testosterone
and DHT specifically but also by other circulating steroid hormones such as adrenal
androgens, estrogen and cortisol et.al due to mutations in the ligand binding domain of
the AR. This relaxation of ligand specificity allows AR to recruit other substitute
androgens when reduced the level of androgen and thus promote the proliferation and
avoid the apoptosis of prostate cancer cells.
LNCaP cells were the first cell line to be identified with this type of mutation in the AR
ligand-binding domain [109]. AR mutations in LNCaP cells extended its ligand binding
with other steroid hormones leading to increasing AR expression and cell proliferation.
A study by Hara et.al demonstrated that long-term culture of LNCaP-FGC cells in
androgen depleted medium with 1µM anti-androgen bicalutamide can produce point
mutants W741C, W741L, and T877A located in AR ligand binding domain [110].
Furthermore,
researches
showed
that
AR
T877A
mutation
allowed
the
hydroxyflutamide (anti-androgen) to trigger AR transcriptional activity and promote
LNCaP cells’ proliferation [111].
Page | 35
Constant with AR mutation described above, many AR mutations found in clinical
cancer cases and associated with progression of prostate cancer and the failure of ADT
but not all androgen independent prostate tumors carried AR mutation. Therefore, there
must be mechanisms other than AR mutations that promote androgen independence.
1.5.2.3 Other pathways
Further studies revealed additional mechanisms including, outlaw AR pathways:
activation of receptor tyrosine kinases resulted in phosphorylation of AR by either the
P13K/AKT pathway or the mitogen activated protein kinase (MAPK) pathway,
producing a ligand independent AR [112]; bypass AR pathway: inhibition of apoptotic
response by activation of anti-apoptotic candidate genes such as BCL-2 which can
promote progression to androgen independence [113]. As prostate cancer cells employ
multiple pathways to survive in reduced level of androgens it is important to identify the
pathways in individual case to design the most efficient therapeutics.
1.5.3 Androgen receptor and growth factors interactions
It has reported that growth factors, such as insulin-like growth factor, fibroblast growth
factor and epidermal growth factor (EGF), can increase AR transactivation under
absence or low levels of androgen [114, 115]. A study by Gregory et.al suggested that
EGF increased level of transcriptional intermediary factor 2/glucocorticoid receptor
interacting protein 1 through mitogen activated protein kinase (MAPK) signalling
pathway; therefore, promote AR transcriptional response in prostate cancer cells [116].
Another angiogenesis growth factor, vascular endothelial growth factor (VEGF), has
Page | 36
been frequently studied recently. The elevated expression of VEGF, which resulted in
promoting vascular growth and the endothelial cell proliferation, was found in
Androgen-stimulated prostate growth [117]. Other reports also showed that functions of
VEGF were activated through hypoxia-induced factor (HIF) regulated by androgens
and/or androgen receptor [118]. Further studies demonstrated the significant correlation
between HIF expression with AR and VEGF expression, and provided firm support for
this control system [119]. Understanding of such cross-talk between AR cascade and
growth factors signalling may provide new opportunities for therapy of aggressive
prostate cancer.
1.6
Angiogenesis
Angiogenesis is a multiple and physiological progress involving the growth of new
capillaries from pre-existing blood vessels. Angiogenesis happens to repair damaged
tissue when wounds are healing; therefore, normal cells can switch on the growth of
blood vessel by releasing angiogenic factors. On the other hand, they also can produce
antiangiogenic factors which switch blood vessel growth off. Tumor angiogenesis is that
cancer cells stimulate the growth of hundreds of capillaries from the nearby blood
vessels, which grow around and/or into the tumor, to establish an independent supply of
nutrients and oxygen. Angiogenesis is not only important to tumor development but also
a critical step in tumor metastasis. The immature, highly permeable blood vessels,
which have little basement membrane and few intercellular junctional complexes,
Page | 37
provide efficient route of exit for tumor cells to leave the primary site, enter the blood
stream, travel to another part of the body and begin to grow there.
1.6.1 Fibroblast growth factors
Fibroblast growth factor (FGF) family is composed of 23 members which are heparinbinding proteins and the size of these single chain peptides is range from 16 to 18 kDa
[120]. FGFs regulate cell functions by binding FGF receptors (FGFR1, FGFR2, FGFR3
and FGFR4) located on the cell surface in the presence of heparin proteoglycans. FGFs
were found to involve various biological functions including cell proliferation, cell
differentiation, angiogenesis and tissue repair. It has been reported that FGF prototypes,
such as FGF-1 and FGF-2, stimulate tumor cell growth rate and organize endothelial
cells to form tube-like structures which results in promoting angiogenesis. In vitro
studies demonstrated that FGF-1 significant increase vascularisation when transfected to
the chicken chorioallantoic membranes (CAMs) and the level of protein kinase B
(AKT) mRNA also enhanced in the experiment indicating the involvement of the AKT
kinase signaling pathway. Moreover, adding the AKT inhibitor significantly reduces
FGF-1 induced angiogenesis in CAMs [121]. Another important heparin binding
prototype of FGF family, FGF-2 has also been correlated with tumor progression by
activation of second of secondary angiogenesis factors, such as vascular endothelial
growth factor (VEGF). Hori et al. provided the evidence that monoclonal antibody
against FGF-2 inhibit tumour growth and vascularization [122]. Other studies also
show that FGF-2 is associated with low differentiation, metastasis and poor survival in
cancer patients [123-125].
Page | 38
1.6.2 Vascular endothelial growth factor (VEGF)
There are at least five members of the vascular endothelial growth factor (VEGF)
family existed in human: VEGF-A (usually referred to as VEGF), VEGF-B, VEGF-C
and VEGF-D and a structurally related molecule, placental growth factor (PlGF). All
members of VEGF family are crucial important angiogenic factors which primarily
target vascular endothelial cells. They promote new blood vessel formation by binding
tyrosine kinase receptors (also known as VEGF receptors) located on cell surface. Three
VEGF receptors have been recognized: VEGFR-1 or fms-like tyrosyl kinase-1(Flt-1),
VEGFR-2 or Kinase insert domain receptor (KDR/Flk-1) and VEGFR-3 or Flt-4. Both
VEGF and VEGF-B bind to VEGFR-1 which is involved in the organization of
development of blood vessel, haematopoiesis and enhance VEGF-induced VEGF-2
signaling during abnormal angiogenesis. VEGF but not VEGF-2 binds to VEGFR-2
results in promoting cell proliferation, mitosis, vascular permeability and angiogenesis,
whereas VEGF-C and VEGF-D are ligands for VEGFR-3, which stimulates
lymphangiogenesis [126].
1.6.2.1 The isoforms of VEGF
VEGF is a ~45kDa homodimeric heparin-binding glycoprotein which was firstly
identified by Senger et al. in 1983. VEGF gene consists 8 exons separated by 7 introns
and is located at chromosome 6p21.3 [127]. So far, there are 12 VEGF isoforms which
have been divided into two families according to their terminal exon (exon 8) splice
site: the proximal splice site, designated as VEGFxxx or distal splice site, designated as
VEGFxxxb [128]. The identified multiple proteins of VEGF expressed in human tissues
and cells are named as VEGF121, VEGF121b, VEGF145, VEGF145b, VEGF165, VEGF165b,
Page | 39
VEGF189, VEGF189b and VEGF206 according to different number of amino acids.
VEGF121 is thought to be the most diffusible isoform due to the absence of a heparinbinding domain. VEGF165, the predominant VEGF isoform secreted by various of cells,
not only exists in diffusible location but also remains bound to cell surface and extra
cellular matrix (ECM). However, the larger isoforms such asVEGF189 and VEGF206
with high affinity to heparin remain localized within ECM (reviewed in [126]).
1.6.2.2 VEGF and prostate cancer
Most of tumor cell lines secrete VEGF in vitro indicating that VEGF may play a crucial
role in tumor angiogenesis. This has been confirmed by in situ hybridization studies
which demonstrated the VEGF mRNA expressed in majority human carcinomas such as
breast cancer, lung cancer, bladder cancer, prostate cancer and ovary caner (reviewed in
[126]). In prostate cancer, VEGF is expressed differently in normal, benign and prostate
cell line. It has been reported that the expression of VEGF mRNA was detected in PIN,
and poorly differentiated tissues, but not in normal prostate tissue [129]. Further studies
also showed that the microvessel density (MVD) was increased significantly in
metastatic prostate cancer sample when compared with non-metastatic prostate cancer
samples[130]. In addition, Du et al. detected the higher expression of hypoxia-inducible
factor (HIF), a key regulator of VEGF expression, in malignant prostate cancer
compared with the normal and benign prostate tissue [131]. Clinical studies revealed
that the level of VEGF expression in serum, plasma or urine was correlated with higher
Gleason grade, metastasis and disease-specific survival [132, 133]. On the other hand,
inhibition of VEGFR-1 and VEGFR-2 using AZD-2171 (Cediranib, AstraZeneca)
induce tumor shrinkage in 56.5% patients (13 out of 23 patients with measurable
Page | 40
disease) with 4 meeting the criteria for partial response [134]. The results above
indicated that VEGF plays crucial role in prostate cancer at both early initiating stage
and later stage for tumor progression and metastasis. VEGF interacts with VEGF-2 to
stimulate endothelial cell proliferation through the mitogen activated protein kinase
(MAPK) pathway and promote vascular permeability, and subsequently with VEGFR-1
to assist the organization of new capillary tubes.
1.6.3 Peroxisome proliferator-activated receptors
Peroxisome proliferator-activated receptors (PPARs) are part of the nuclear receptors
family of transcription factors and originally identified in Xenopus frogs as receptors
that induce the proliferation of peroxisome in cells in the early 1990s [135]. The
functional role of these ligand activated transcription factors is involved in regulating
fatty acid catabolism, lipid storage and glucose metabolism in the body. There are three
isoforms of PPARs: PPARα, PPARβ/δ and PPARγ. They are expressed in various
tissues and play different roles.
Page | 41
1.6.2.1 Structure of PPAR
Despite the location and functional differences, PPAR isoforms share similar structure
and functional domains. Like other nuclear receptors, PPARs are composed of four
major domains: A/B, C, D and E/F domains (Figure 1.9).
Figure 1.9
The schematic view of the domain structure of PPARs.
The N-terminal region contains PPARs A/B domains which are different in both length
and amino acid sequence. The A/B domain contains a transactive domain independent
of the presence of the ligand, which is termed activation function 1 (AF1). The activity
of this domain can be regulated by protein kinase phosphorylation [136]. The C domain,
which is the most conserved region, consists of a DNA binding domain (DBD). This
domain contains two typical zinc finger motifs which bind to specific sequences of
DNA and responsible for binding PPAR to peroxisome proliferator response element
(PPRE) within the promoter region of the target gene. The D domain is a highly flexible
hinge region. The C-terminal region of PPAR is E/F domain which contains ligand
binding domain (LBD) and ligand dependent activation domain (AF2). The LBD of
PPARs consists 13 α helices and a β sheet forming a hydrophobic pocket which is about
twice larger than other nuclear receptors [137]. This region is important for activation of
PPAR binding to the PPRE and also plays an important role in nuclear localization. In
addition, the researches demonstrated that PPARs could be activated by a variety of
Page | 42
fatty acid at a certain concentration [138]. PPARs form heterodimers with the 9-cis
retinoic acid receptor (RXR), which result in a conformational change of both receptors,
allowing the heterodimers to bind to the PPRE DNA promoter region containing repeat
motifs (AGGTCANAGGTCA) and regulate the gene transcription (Figure 1.10).
Figure 1.10
Model for PPARs signalling pathway.
1.6.2.2 PPAR β/δ
PPAR β/δ is expressed ubiquitously throughout the body with different levels in certain
cell types. PPAR β/δ can be activated by natural ligands, such as fatty acids, retinoid
acid and prostaglandins as well as synthetic ligands including GW501516, GW0742 and
L165041. Several studies demonstrate that PPAR β/δ plays important roles in cell
proliferation, differentiation and apoptosis. Activation of PPAR β/δ can stimulates the
cell proliferation in colon cancer, liver cancer, lung cancer and breast cancer cell lines
(reviewed in [139]). It has also been reported that PPAR β/δ increases cell proliferation
in PNT and LNCaP but not in DU145 and PC-3 prostate cancer cell lines [140].
Page | 43
However, the result data on the role of PPAR β/δ in cell proliferation are inconsistent
and controversial. In addition, a number of recent studies suggest that activation of
PPAR β/δ with GW501516 also can stimulate angiogenesis by enhancing the expression
of VEGF [141, 142]. Strong evidence suggests that ligand activation of PPAR β/δ can
enhance the expression of 3-phosphoinositide dependent protein kinase 1 (PDK1),
subsequently activation of the downstream protein kinase: protein kinase B (AKT) by
phosphorylation. Activation of PDK1/AKT signalling pathway has a direct effect on
cell apoptosis through phosphorylation of the BCL-2 family member BCL-2 associated
death promoter (BAD) thereby suppressing cell apoptosis and promoting cell survival.
Recent work by Schug et al. also supports this PDK1/AKT anti-apoptosis signalling
pathway regulated by PPAR β/δ [32].
1.6.2.3 PPAR γ
PPARγ has been the most extensively studied isoform of PPAR family. It is mainly
expressed in adipose tissues and plays a crucial role in adipocyte differentiation and
lipid metabolism. Previous observation showed PPARγ ligands promote the VEGF
production in bladder and prostate cancer cells [143, 144]. Analogous findings were
also reported by Cho et al. who detected a significant increase in VEGF and its
receptors in epithelial cells treated with PPARγ synthetic ligand, thiazolidinediones. In
addition, activation of PPARγ also stimulates the tumor formation in colon cancer
[145]. On the other hand, there are several lines of evidence to support that PPARγ
ligand activation are capable of inhibiting angiogenesis progression under certain
conditions. It is suspected that the various time and dose of PPARγ ligand treatment or
the variety of organisms and cells examined may lead to such discrepancies.
Page | 44
1.7
Fatty acid binding proteins
1.7.1 Fatty acid binding protein family
As mentioned in previously in 1.1.3.3, several studies highlighted a possible association
between high incidence of clinical prostate cancer in western developed nations and
high dietary fat intake. Fatty acids, which are produced by hydrolysis of ester linkage in
oil or fat, commonly have a chain of 4 to 28 carbons (short or medium chain: aliphatic
tails are less than 12 carbon atoms; long chain: aliphatic tails are between 12 and 24
carbon atoms). Fatty acids not only function as an energy source but also act as cell
signalling molecules which regulate gene expression, proliferation and apoptosis
pathways [146]. Fatty acids are very soluble once they secreted from fat cell to the
aqueous cytoplasm environment. Then, free fatty acids cross the plasma membrane
either by passive diffusion or though transporters. Once fatty acids reach the cytoplasm,
they either enter a metabolic pathway or bind to intracellular fatty acid binding proteins
which carry them to different organelles.
Fatty acid binding proteins (FABPs) are a family of cytoplasmic proteins that reversibly
bind to long chain fatty acids and other lipophilic substances such as eicosanoids and
retinoids, with high affinity [147]. FABP super family can be classified into two groups:
(1) FABPpm, located in the plasma membrane; (2) FABPc (usually referred to as
FABPs), intracellular or cytoplasmic proteins. Since the initial discovery of FABPs in
1972, so far nine members of FABP have been identified as shown in Talbe 1.2.
Page | 45
Table 1.2
Family of fatty acid binding proteins [146]
Page | 46
FABPs are small intracellular proteins with similar molecular masses around 15kDa.
Members of FABP family display various pattern of tissues expression. Although
FABPs were named according to the tissue in which they were first identified, none of
members is specific for one tissue or cell type and some tissues express more than one
FABP, which indicates that FABPs may have unique functions. Some of FABPs
expressed in few specific tissues but some of them exhibit broad tissue distribution. For
instance, LiverFABP (FABP1) is highly expressed in the liver and is also found in the
intestine, pancreas, kidney and lung. Whereas E-FABP (FABP5), which is one of the
most ubiquitously expressed FABP is widely expressed in the skin, tongue, adipocyte,
macrophage, dendritic cell, mammary gland, brain, intestine, kidney, liver, lung, heart,
skeletal muscle, testis, retina, lens and spleen [146]. Expressions of FABPs are
significantly higher in hepatocytes, adipocytes and myocytes, where fatty acids are
prominent substrates for lipid biosynthesis, storage, or breakdown compared to amounts
found in other tissues [148]. It has been reported that increasing fatty acid also elevated
the level of FABP expression in most cell types [149]. These observations indicate that
FABPs response to increased fatty acid concentration and trigger the downstream
signalling pathways regulating cells behaviours.
1.7.2 Structures of fatty acid binding proteins
The structures of FABPs have been investigated by X-ray crystallography, nuclear
magnetic resonance and other techniques. The members of FABPs have an abroad range
of sequence diversity varying from 17-67% [150]. Despite the differences between
FABPs sequences, these proteins show a great similarity in their three-dimensional
structures as shown in Figure 1.11.
Page | 47
Figure 1.11
Three-dimensional structures of fatty acid binding proteins [146]
Ligand-binding FABPs are shown above (The figures were created using
PyMOL). (a) FABP1 (Protein Data Bank (PDB) code: 1lfo); (b) FABP2
(PDB code: 2ifb); (c) FABP3 (PDB code: 2hmb); (d) FABP4 (PDB
code: 2ifb); (e) FABP5 (PDB code: 1b56); (f) FABP8: (PDB code:
1pmp); (g) FABP6: (PDB code: 1o1v); (h&i) FABP7: (PDB code: 1fe3
& 1fdq).
Page | 48
All members of FABPs form a β barrel structure, which consists of two orthogonal β
strands with five antiparallel β sheets in each strand. The fatty acid binding site is
located inside the β barrel structure capped by helix-loop-helix motif which also
connects the two β strands [151, 152]. Within the β barrel structure is a large cavity
lined with hydrophobic and polar amino acids [147]. Most FABPs bind only a single
fatty acid which is oriented inwards and anchored in the cavity through the interaction
with tyrosine and 2 arginine residues [153]. The conserved fingerprint, providing a
signature for all FABPs, has recently been revealed as shown in Figure 1.12 (Prints
pattern FATTYACIDBP; PR00178).
Page | 49
Figure 1.12
Fingerprint for fatty acid binding proteins [146]
Three highly conserved motifs are presented with the ribbon diagram.
FATTYACIDBP1 (blue ribbon) forms part of first β strands (βA). It also
includes a nuclear localization signal (NLS) and a hormone-sensitive
lipase (HSL) binding site. The nuclear export signal (NES) domain is
located in FATTYACIDBP2 (green ribbon) which includes β sheet 4 and
5 (βE). FATTYACIDBP3 (red ribbon) encodes β sheet 9 (βI) and 10
(βJ). The key amino acids are also marked with blue, green and red
respectively.
Page | 50
1.7.3 Functional role of fatty acid binding proteins
Various functions have been proposed for intracellular FABPs which increase the
solubility of fatty acids and facilitate the transport of them to specific organelles, such
as to the lipid droplet for storage; to the endoplasmic reticulum for signalling and
membrane synthesis; to mitochondria or peroxisome for oxidation; to cytosolic to
regulate enzymes activity (reviewed in [146]). Numerous studies also found that FABPs
appear to be involved in regulating nuclear transcription factors (PPARs) activities
through ligand-dependent translocation to the nucleus. The functional roles of FABPs in
intracellular fatty acids are shown in Figure 1.13.
Figure 1.13
The functions of FABPs in the cell [146]
Page | 51
Growing evidence suggests that FABPs bind to fatty acids and interact directly with
PPARs. Further studies also detected the presence of FABPs in the nucleus of different
cell types, such as hepatocytes, heart myocytes, cancer cells and cell lines transfected
with FABPs using immunofluorescence imaging or imaging of living cells expressing
GFP-tagged FABPs [154, 155]. In addition, Tan et.al revealed that C-FABP located into
the nucleus in presence of PPARβ, whereas A-FABP undergoes a nuclear localization in
response of ligand for PPARγ but not PPARβ [156]. These results indicated that
individual FABPs may have unique signalling pathway in nucleus to control the specific
gene expression.
1.7.4 Cutanious fatty acid binding protein in prostate cancer
Cutanious fatty acid binding protein (C-FABP), also known as FABP5, E-FABP and
K-FABP, is a typical member of FABP family with a molecular weight of 15kDa. CFABP was originally isolated from psoriatic skin and located at chromosome 8q21.13.
Similar to other FABP members, it binds with high affinity to long chain fatty acid.
Our previous studies identified and characterized a metastasis-inducing gene, which is
coding for the C-FABP using systematic differential display [157]. Jing et.al also
showed that C-FABP is differentially expressed in normal, benign and malignant breast
and prostate cell lines and capable to develop metastasis in vivo when transfected CFABP into Rama37 cells which are non-metastatic rat mammary cells [157]. Further
investigation on the possible down-steam mediator demonstrated that overexpression of
C-FABP significantly increases the expression of VEGF, a crucial factor for tumor
growth and expension and also elevated the microvessel density of the primary
Page | 52
tumor [158]. The immunocytochemical analysis detected that the level of C-FABP
expression is associated with prostate cancer malignant progression. However, no
relation between expression level of C-FABP and degree of malignancy has been found
as C-FABP expression is not greatly different in between low and high Gleason grades
[159]. Suppression of C-FABP expression by antisense transfection in PC-3M cells
decreased the invasive capacity in vitro and reduced the tumorigenicity in vivo.
Moreover, the suppression of VEGF expression has also been detected in these
transfected cell lines [159]. The results above indicate that overexpression of C-FABP
may stimulate the expression of VEGF and subsequently promote the angiogenesis to
facilitate tumor formation and metastasis. Recent studies by Morgan et.al also
confirmed that suppressing the expression of C-FABP in PC-3M cells using siRNA
silencing technique resulted the significant reduction of tumorigenicity both in vitro and
in vivo [160]. However, how exactly C-FABP elevates the expression of VEGF is still
unclear.
Page | 53
1.8
Aim of this thesis
 To investigate whether the fatty acid binding activity is essential for CFABP to promote the tumorigenicity of prostate cancer cells transfected with
C-FABP
 To examine biological behaviours and the fatty acid transporting activity in
prostate cancer cells before and after transfected with C-FABP and its
mutants
 To study whether the elevation of C-FABP expression in transfectants
increases the expression level of VEGF
Page | 54
Chapter 2 Materials and Methods
2.1 Materials
2.1.1 Materials for cell culture
Reagents
Supplier
Cell culture plates and filter cap flasks
Nunc
Labofuge 400R (Centrifuge)
Heraeus
Cryogenic vials
Nunc
DMSO
Sigma
EndoGRO basal medium
Millipore
EndoGRO LS supplement kit
Millipore
Eppendorf tubes (1.5ml)
Sarstedt
Fetal calf serum
Biosera
Fluorescence microscope
GeneJammer transfection reagent
Stratagene
Geneticin (G418)
PAA
Hemocytometer slide
Weber scientific international
Hydrocotisone
Sigma
Incubator (Cell culture)
Thermo Scientific
L-Glutamine
BioWhittaker
Penicillin/Streptomycin
BioWhittaker
Phosphate buffered saline (Tablet)
Gibco
RPMI 1640
PAA
Page | 55
Reagents
Supplier
Sodium pyruvate
Sigma
Testosterone
Sigma
Tissue culture pipettes (5ml – 25ml)
Greiner bio-one
Trypsin
Gibco
Universal tube (25ml)
Greiner bio-one
Versene
Gibco
Water bath
Grant Instruments
2.1.2 Materials for fatty acid uptake assay
Reagents
Supplier
BSA
Sigma
BODIPY 558/568C12
Invitrogen
EPICS XL Flow Cytometer
Beckman Coulter
2.1.3 Materials for cell proliferation assay
Reagents
Supplier
96-well plate
Nunc
MTT
Sigma
Multiscan plate reader
Labsystem
Page | 56
2.1.4 Materials for cell invasion assay
Reagents
Supplier
24-well Boyden chamber plate
Boyden chamber
Crystal violet
Matrigel
BD
2.1.5 Materials for soft agar assay
Reagents
Supplier
GelCount
Oxford Optronix
Low melting point agarose
Sigma
2.1.6 Materials for Tumorigenicity in vivo
Reagents
Supplier
Balb/C immuno-incompetent nude mice
Matragel
Needles and Syringes
BD
Page | 57
2.1.7 Materials for Histology
Reagents
Supplier
Formalin
Tissue-Tek VIP
Sakura
Embedding cassette
Microtome HM355
Mirrom
Stainless steel disposable blade
Superfrost microscope slides
Menzel-Glaster
Sodium citrate
Sequenza Slide Rack
Tween-20
Sodium chloride
Tris-base
Bovine serum albumin
Coverslip (20x40mm)
2.1.8 Materials for measurement of VEGF
Reagents
Supplier
Sodium carbonate
3, 3’, 5, 5’-Tetramethylbenzidine
Recombinant human VEGF
Phenol red-free RPMI 1640 medium
Page | 58
Charcoal stripped FCS
VEGF microplate
Sulfuric acid
HRP-Streptavidin solution
Wash solution
Biotinylated secondary antibody
2.1.9 Materials for in vitro angiogenesis assay
Reagents
Supplier
In vitro angiogenesis assay kit
Human umbilical vein cells
EndoGRO basal medium
EndoGRO LS supplement kit
Crystal violet
2.1.10 Materials for general molecular biology
Reagents
Supplier
Agarose
DEPC
Ethanol
Microcentrifuge
Sodium acetate
Page | 59
Bromophenol blue
Xylene cyanol FF
Glycerol
Ethidium bromide
NanoDrop ND-1000
NanoDrop Technologies
Restriction enzymes
T4 DNA ligase
2x quick Ligation buffer
2.1.11 Materials for transformation of competent bacteria
Reagents
Supplier
DH5 E.coli bacteria
Bactotryptone
Yeast extract
NaCl
KCl
LB agar
MOPS
CaCl2
Glycerol
MgCl2
MgSO4
Glucose
Page | 60
Ampicillin
Kanamycin
LB broth
2.1.12 Materials for RT-PCR
Reagents
Supplier
10x PCR buffer
Taq DNA polymerase
dNTPs
Thermocycler
DTT
Oligo (dT)20
RNaseOUT
5x first strand buffer
SuperScriptIII reverse transcriptase
2.1.13 Materials for Western blot
Reagents
Supplier
CelLytic-M
Sigma
Roller mixer
Dye reagent
Bio-Rad
Bradford reagent
Page | 61
Nitrocellulose membrane
Coomassie blue
Protoblock reagent A &B
ACTG (National diagnostics)
Page | 62
2.2 Methodology
2.2.1 Cell culture
2.2.1.1 Routine cell culture
All routine cell culture manipulations took place in a tissue culture hood located in the
Tissue Culture Laboratory. Cells were cultured in 75cm2 cell culture flasks and
maintained at 37oC in a humidified incubator which contains 5% (v/v) CO2. LNCaP
cells were cultured in RPMI1640 nutrient medium supplemented with 10% (v/v) Foetal
calf
serum
(FCS),
2mM
L-glutamine,
1mM
Sodium
pyruvate,
100IU
penicillin/streptomycin (PEN-STREP), 5µg/ml 5-dihydroxytestosterone and 5µg/ml
hydrocortisone. HUVEC cells were cultured in EndoGRO basal medium (EBM) with
EndoGRO LS supplement kit. For transfected cell lines, geneticin (G418) was added to
the previous components gave the final working concentration of 1mg/ml. Both routine
and experimental culture media were replaced every three days.
2.2.1.2 Thawing of cells
Cryogenic vials of cells were defrosted in a water bath at 37oC and immediately
transferred into a universal tube with 20ml of complete cell culture medium. The cells
were then pelleted by centrifugation at 900rpm lasting for 3 minutes. Then, the
supernatant was discarded and the pellet was resuspended in a suitable volume of
complete medium contained with 10% FCS. Finally, cells were plated out in a 75cm2
cell culture flask and maintained within a controlled atmosphere of 5% CO2 at 37oC.
Page | 63
2.2.1.3 Sub-culture of cell lines
Cells that had grown to approximately 60-80% confluence were passaged to allow
continuous growth. Medium was completely removed from the flasks. Then, growing
cells were washed by phosphate buffered saline (PBS) and incubated at 37 oC with 3.5ml
of 2.5% (v/v) trypsin/versene solution for a period 6 minutes or until the cells had
rounded up and were starting to detach from the culture dish. The trypsin/versene
solution was inactivated by adding 10 ml of complete medium. The cells were then
transferred to a universal tube and centrifuged at 900rpm for 3mins. The supernatant
was removed and cells were resuspended in cell culture medium. The cells were
aliquotted at the required concentration and maintained within a controlled atmosphere
of 5% CO2 at 37oC.
2.2.1.4 Counting cells
The number of cells was calculated by using an improved Neubauer double counting
chambers hemocytometer. Cells were detached as described previously. Mix cell
suspension thoroughly and load 10µl of cell suspension to a counting chamber of
hemocytometer with 9 (3x3) squares. Then, count cells in four corner squares under the
microscope. The total number of cells is calculated using the equation listed below:
Total number of cells = Average cell count per square x Dilution x 104 x Total volume (ml)
Page | 64
2.2.1.5 Freezing cells
Cells were selected for freezing when they were approximately 60-80% confluence.
Before freezing, the cell culture medium was replaced with fresh medium for 24 hours.
The cells were detached as described in 2.2.1.3 and resuspended in 10ml of completed
medium. Then, the total number of cells was counted as mentioned in 2.2.1.4. The cells
were re-centrifuged at 900 rpm for 3 minutes. The supernatant was removed and the cell
pellet was broke up by repeated pipetting with freezing medium (Complete medium
with 7.5% (v/v) DMSO) to give a concentration of 1x106 cells/ml. Aliquots (1ml) of the
cell suspension were pipetted into cryogenic vials and placed into a nalgene
cryo-preserver box containing 250ml of isopropyl alcohol. The box stored in a freezer at
–80oC overnight before cryogenic vials transferred for long term storage in liquid
nitrogen.
2.2.2 Cell transfection
2.2.2.1 Stable transfection of the LNCaP cell line using GeneJammer
A polyamine based method of transfection (GeneJammer) was used to insert
pIRES2-EGFP into LNCaP cells. Prior to transfection, LNCaP cells (2x105) were seeded
in a 6-well tissue culture plate and grew the cells overnight in complete LNCaP culture
medium to reach 50-70% confluence. In the mean time, 97µl of sterile, room
temperature (RT), serum-free, antibiotic-free RPMI medium was transferred to a 1.5ml
eppendorf tube and added with 3µl GeneJammer transfection reagent. After incubation
for 5mins at RT, 2µg of DNA was added to the diluted GeneJammer transfection
reagent and mixed gently. The mixture was incubated at RT for 30 minutes and added
dropwise to the wells of the 6-well tissue culture plate. Then, the transfection mixture
Page | 65
was distributed evenly by rock the plate back and forth. The control transfections were
also performed as shown in Figure 2.1. After incubation in standard growth conditions
for 48 hours, cells were split into 5 separate 9cm cell culture plate and cultured in
selective medium containing 0.5mg/ml Geneticin for 10 days. The Geneticin was
removed and the surviving transfected clones were allowed to recover in routine
medium for one week.
Figure 2.1
Three mock-transfection controls were performed by putting cells
through the transfection procedure without adding GeneJammer
transfection reagent or DNA or both of them.
Page | 66
2.2.2.2 Transfected clone selection by cell image
Cells were then re-cultured in selective medium for a further 3-4 weeks until the cells in
control transfection died out and healthy cell clones had formed in the cell culture plate.
As pIRES2-EGFP vector (as presented in Figure 2.2) contained the enhanced green
fluorescent protein (EGFP) coding region, successfully transfected cell colonies with
the expression of fluorescent marker were detected under a fluorescence microscope.
Because the proportion of cells transfected within a population is relatively low (less
than 10%), the ring cloning methodology was used to isolate colonies. For each batch
transfectants, 5 colonies with best expression of EGFP were marked on the down-side
of 9cm plate for ring cloning.
Figure 2.2
Circular maps and lists of features for the pIRES2-EGFP vector
Page | 67
2.2.2.3 Ring cloning of transfected cells
The tops of 1ml pipette tips were cut to larger the cloning rings which were sterilised
with silicon grease and forceps by autoclaving. The selective medium from the
transfectant plates was removed and the plates were washed with PBS. Using forceps,
the cloning rings were dipped in silicon grease and placed over the colonies. The small
amount of silicon grease allowed the rings to stick to the plate and formed a watertight
seal around the colony. Then, 100µl of 2.5% (v/v) trypsin/versene solution was added to
isolated colonies and incubated for 6 minutes or until the cells were rounded-up. The
detached cells were transferred to a 1.5ml eppendorf tube and the trypsin inactivated by
adding an equal volume of routine cell culture medium. The cells were centrifuged for 3
minutes at 900rpm. After remove of supernatant, the cell pellet was resuspended in 1ml
selective culture medium containing 0.5mg/ml geneticin and plated into a 24-well plate
and maintained within a controlled atmosphere of 5% CO2 at 37oC.
2.2.3 Fatty acid uptake assay
Fatty acid uptake assay is designed in order to test long chain fatty acid (LCFA) uptake
kinetics in different transfectant cells containing fatty acid transporters. Conventional
protocols utilizing radioactivity often require cell lysis and processing at very low
temperature. Whereas, uptake of fluorescently-labeled fatty acids requires neither
radioisotope usage nor cell lysis and also the fluorescence intensity in each individual
cell can be measured by flow cytometry precisely. The red fluorescence-labeled LCFA,
BODIPY 558/568C12 (4, 4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene-3dodecanoic acid), was used as the analogue of natural LCFA in the fatty acid uptake
assay. BODIPY analogue behaves much like natural fatty acids: it becomes activated by
Page | 68
acyl-CoA attachment; is incorporated into triglycerides; and accumulates in intracellular
lipid droplets. In addition, the BODIPY analogue is also known as the substrate for fatty
acid transporters since its uptake by adipocytes and can be competed by natural LCFA.
Cultured cells (1x105 cells) in 2ml routine culture medium were seeded into 6-well
plates and incubated at 37oC, 5% CO2 overnight. After replacing the medium with 2ml
of solution containing 25μg BODIPY 558/568C12 in 200μm BSA/PBS, the cells were
incubated for 30mins at 37oC, 5% CO2. The fatty acid uptake was stopped by removal
of BODIPY fatty acid solution followed by addition of 3ml of an ice-cold stop solution
(PBS containing 0.5% BSA). The stop solution was discharged after 2 min and the
culture plates were washed another two times by fresh ice-cold stop solution. Then,
cells were detached using 2.5% (V/V) trypsin/versene at 4oC and the fluorescence
intensity of each cell line was measured with an EPICS XL Cytometer at the wave
length of 570 nm to assess the fatty acid uptakes.
Page | 69
2.2.4 Cell proliferation assay
2.2.4.1 Preparation of growth curve
Cells were grown to 60-80% confluence in 175cm2 flasks and harvested as previously
outlined and resuspended in 10ml of complete culture medium. Each cell line was
counted using a hemocytometer as previously mentioned in section 2.2.1.4 and was
made up in 5x105/ml in 4mls of complete culture medium. A standard growth curves
was prepared in serial dilution at: 6.25x103/ml, 1.25x104/ml, 2.5x104/ml, 5x104/ml,
1x105/ml, 2.5x105/ml and 5x105/ml. Then, 200µl of cell suspension from each dilution
was plated into a 96-well plate in triplicate. Each cell line requires its own standard
curve.
In the mean time, the cells for proliferation assay were also prepared at the
concentration of 5x104/ml. Similar as preparation of standard curve, 200µl cell
suspension containing 1.25x104 cells was plated into five separate 96 well plates in
triplicate. After overnight incubation with 5% CO2 at 37oC, the cell growth of the
standard curve and first experimental day analysed by the MTT assay as described in
section 2.2.4.2. The cell proliferation assay was set to run for five days at each plate per
day.
2.2.4.2 Assessing cell numbers using MTT assay
MTT is a yellow coloured chemical and it entered into the mitochondria of the cells. In
mitochondria, MTT is oxidised into an insoluble blue dye known as formazan which
forms blue crystals in the cytoplasm. The crystals are dissolved by adding DMSO and
leave a coloured liquid, which is directly proportional to the number of cells present.
Page | 70
The optical density (OD) of the cell population colour is measured accurately with a
Multiscan plate reader at 570nm. For each cell line, a standard curve was constructed by
plotting OD (570nm) against the number of cells. The cell number of the test samples
was determined by extrapolating from the standard curve.
MTT stock solution is prepared at a concentration of 5mg/ml (100mg MMT in 20ml
PBS) and stored at 4oC. At each time point (every 24 hours), 50l of MTT stock
solution was added to each well in a 96-well plate and the cells were incubated at 37oC,
5% CO2 for 4 hours. The cell culture medium and MTT is removed gently to avoid
disturbing cells followed by adding 200l of DMSO to each well including blank
control wells mixed by pipetting up and down. The plate was incubated for a further 10
minutes at 37oC, 5% CO2 and read at 570nm in the optical density plate reader. The
growth rate for each cell line is quantified against its own standard curve to obtain the
number of cells/well in each day for five days. Graphs of cell number/time were plotted.
2.2.5 Cell migration assay in Boyden chamber system
The Boyden chamber system was used to examine the difference in invasive ability in
cell lines expressing wild-type and mutant C-FABPs. The results are often variable, as
many factors are involved in the assay, including the number of cells added to the upper
chamber, the duration of the assay and the way to measure the number of the cells
[161]. Therefore, it is important for the system gives reproducible results by setting up
an optimized procedure considered the variable mentioned above. All materials used in
the section can be found in section 2.1.
Page | 71
Cells to be used in invasion assay were cultured in flasks until 60-80% confluent. Cell
invasion assays were carried out using a modified Boyden chamber system. Wells of the
24-wells plate were divided into upper and lower compartment by 6.5mm diameter
polycarbonate membrane containing 8µm pores. The polycarbonate filters were wetted
with 200μl RPMI medium for 30mins and the medium was removed followed by
adding further 100μl RPMI medium containing 30μg of matrigel to the upper side of the
filters. The plates were incubated at 37oC, 5% CO2 for 2 hours to allow the matrigel
solidification. The assays were set up with 1x105 cells in the upper compartment in
200μl of 2% (v/v) FCS growth medium and 500μl of 10% (v/v) FCS growth medium
was placed to the lower chamber. After 24 hours incubation at 37oC, 5% CO2, the cells
remaining in the upper side of the filters were removed gently by cotton swabs and the
cells attached to the lower side of the filter were fixed and stained by crystal violet
reagent. None of cell was found in lower compartment indicated that all cells invaded
through the matrigel coated filter were attached to the lower side of the filter. The
numbers of the invaded cells were counted under microscope. The results were finally
normalized by the cell number in the 24-well plate after 20 hours incubation because the
cells after typsinisation may vary in viability. In this way, the cell motilities measured
from different cell lines were more comparable. The results are the mean ± SD of three
separate experiments.
Page | 72
2.2.6 Soft agar assay
Soft agar assay is designed in order to examine the tumorigenicity of each cell lines in
anchorage-independent environment. The assay was carried out in 6-well plates which
were pre-coated with 2ml of 2% (w/v) low melting point agarose in routine culture
medium with 10% (v/v) FCS and the mixture solidified in refrigerator at 4oC for
20mins. The cells were routinely grown to 60-80% confluent in flasks, detached and
counted as previously described in section 2.2.1.3 and 2.2.1.4. Then, the cells were
washed once by centrifugation and resuspended in routine culture medium with 10%
(v/v) FCS at the concentration of 1x104/ml. The cell suspension was mixed with 2% low
melting point agarose in a ratio of 1:1 and 2ml of mixtures were seeded to the precoaded wells to make the final cell concentration at 1x104 per well. The plates were
placed at 4oC until solidified. Once the top layer was set, the 6-well plates were placed
in the incubator at 37oC, 5% CO2 for 4 weeks. During this period, 2 drops of routine
culture medium were added to each well twice a week to keep the soft agar moist. At
the end of the soft agar assay, colonies were dyed by adding 2ml of MTT (5mg/ml)
followed by incubation at 37oC, 5% CO2 for 4 hours. Colonies larger than 150μm in
diameters were counted by the GelCount.
Page | 73
2.2.7 Tumorigenicity in vivo
The in vivo tumorigenicity of the transfectant lines were explored using Balb/C
immuno-incompetent nude mice (4-6 weeks old). The experiments were carried out
using four groups of mice (8 animals in each group) to assess the level of
tumorigenecity in LNCaP cells transfected with wild-type C-FABP, two mutant
C-FABPs and pIRES2-EGFP vector only. All Balb/C immuno-incompetent nude mice
were housed in the University of Liverpool animal unit and experiments were conducted
in accordance to UKCCCR guidelines under Home Office Project Licence PPL 40/2963
to Professor Y. Ke.
The cells for the in vivo tumorigenecity assay were grown up to ~80% confluence in
175cm2 flasks and harvested as described earlier in section 2.2.1.3. Then, the cells were
centrifuged at 900rpm for 3mins and resuspended in PBS at the concentration of
1x107/ml. The cell suspension was mixed with matrigel in a ratio of 1:1 and kept on ice
during transport to the animal unit. The cells were inoculated subcutaneously into right
flank of the nude mice at the density of 2x106 cells in 200l PBS/matrigel (1:1) mixture
using a 1ml syringe. Tumor volume was measured every three days and calculated by
the following formula: Length x Width x Height x 0.5236 [162] and the primary tumour
measurement was taken by weighing the tumour at autopsy.
Page | 74
2.2.8 Histology
2.2.8.1 Dissection of nude mouse tissues
The nude mice were examined every three days and autopsy after 60 days or when
primary tumor volume exceed one third of the body size of nude mice. Samples of the
primary tumor, liver, pancreas, lungs, kidneys, lymph nodes and any other tissues of
abnormal appearance were removed from the animals and fixed in 10% neutral buffered
formalin (NBF) for 24 hours before processed routinely for histological evaluation.
2.2.8.2 Embedding in paraffin wax and sectioning
The fixed tissue samples were trimmed and placed within an embedding cassette and
processed on a Tissue-Tek VIP5 processor. The processed tissue was then embedded in
paraffin wax at 60οC and cooled on ice. After solidification, the blocks were sectioned
at RT on a Microm HM355 microtome using a microtome knife containing a stainless
steel disposable blade. Sections were cut to 4μm thickness, placed on 3-aminopropyl
triethoxy-saline (APES) coated Superfrost microscope slides and incubated at 37οC
overnight prior to processing with Haematoxylin and Eosin staining using a Varistain
24-4 automated stainer or immunohistochemistry.
2.2.8.3 Immunohistochemistry
Immunohistochemistry is a technique that widely used in research to understand
distribution and localization of biomarkers and differentially expressed proteins in
different parts of a tissue and it enables the localization and visualization of tissue
antigens in-situ by the use of specific antibodies. Firstly, specific antibodies are applied
to bind to the tissue antigen. Secondly, antibody-antigen complex is bound by a horse
Page | 75
radish peroxidise (HRP)-conjugated secondary antibody. Finally, the enzyme-labeled
complex can be visualized by the use of the 3, 3’-Diaminobenzidine (DAB) which
produces a brown-colored precipitate in the presence of HRP. The step-by-step
immunohistochemical staining procedures are listed in Table 2.1.
Table 2.1
Step 1
Step 2
The immunohistochemical staining procedures
The mounted sections were de-waxed in two 5mins changes of xylene.
Shake off excess liquid and dehydrate slides in two changes of fresh
absolute ethanol for 1min each.
Step 3
Slides were incubated in 3% (v/v) hydrogen peroxide/methanol for
12mins to quench endogenous peroxidise activity.
Slides were rinsed in running water for 3mins and antigen retrieval was
Step 4
carried out by microwaving tissue sections for 15mins at full power in
10mM sodium citrate buffer (pH 7.6).
Step 5
Slides were allowed to cool down for further 15mins and placed in
running water before being racked into sequenza slide rack.
1. Slides were washed twice by Tris-Buffered Saline Tween-20 (TBST) and
Step 6
incubated with primary antybody in 200µl of 1% Bovine serum albumin
(BSA)/TBST for 1 hour. Negative controls were incubated with
BSA/TBST alone for the same period of time.
Step 7
Slides were washed three times using TBST and incubated in 200 µl of
Envision FLEX/HRP for 20mins.
Slides were washed three times using TBST and incubated in 200 µl of
Step 8
EnvisionTm FLEX DAB+ chromogen mixed with EnvisionTm FLEX
substrate (1dop/ml) for 20mins.
Page | 76
Step 9
Slides were removed from sequenza slide rack and washed in running tap
water for 1 min.
Step 10
Slides were counterstained in Haematoxylin solution for 50 seconds and
washed briefly in running tap water.
Step 11
Slides were dipped in 1% acid alcohol for five times and washed briefly
in running tap water.
Step 12
Slides were incubated in Scott’s tap water blueing agent for 30 seconds
and washed thoroughly in running tap water.
Step 13
Slides were dehydrated in five changes of absolute methanol (30 seconds
each change).
Step 14
Slides were cleared of any remaining water by incubating in two changes
of Xylene (30 seconds each change).
Step 15
Slides were mounted with cover slips (20x40mm) using DPX synthetic
resin.
Step 16
The stained sections were observed by a light microscope and graded as
negative, weakly positive, moderately positive and strongly positive.
Page | 77
2.2.8.4 The scoring system used for immunohistochemical staining
The immunohistochemical scoring system used in this study was a method devised by
Remmele et.al [163]. The intensity and distribution of positive staining was evaluated
two score system by three observers.
The first evaluation employed a four-point scale for intensity with slides being scored as
0, 1, 2 and 3 as listed below:
0 = no positive staining observed
1 = predominantly weak staining observed
2 = predominantly moderate staining observed
3 = predominantly strong staining observed
Eight random fields of each slide were counted at x40 magnification according to a
positive control sample and a negative control sample.
The second evaluation employed a five-point scale for distribution with slides being
scored as 0, 1, 2, 3 and 4 as listed below:
0 = Less than 10% of the cells positively stained
1 = 10-29% of the cells positively stained
2 = 30-49% of the cells positively stained
3 = 50-79% of the cells positively stained
4 = 80-100% of the cells positively stained
The scores from both intensity and distribution were multiplied to give a final score for
grading which is listed below:
(-) = 0 (negative expression of the protein)
(+) = 1-4 (weak expression of the protein)
(++) = 5-8 (moderate expression of the protein)
(+++) = 9-12 (strong expression of the protein)
Page | 78
2.2.9 Immunosorbent assay for the measurement of VEGF
The quantitative measurements of human VEGF expression in LNCaP transfectants
were carried out using the RayBio human VEGF ELISA (enzyme-linked
immunosorbent assay) kit. This ELISA assay employs an antibody specific for human
VEGF coated on a 96-well plate. Standards and samples are pipette into the wells and
VEGF is bound to the wells by the immobilized antibody. The well are washed and
incubated with biotinylated anti-human VEGF antibody. After washing away unbound
biotinylated antibody, the HRP-conjugated streptavidin is added to bind to the
secondary antibody. Finally, 3, 3’, 5, 5’-Tetramethylbenzidine (TMB) is pipetted to the
wells and color develops in proportion to the amount of VEGF bound.
2.2.9.1Preparation of standard curve
The recombinant human VEGF was diluted to a concentration of 18,000pg/ml standard
solution in a diluent, 50mM sodium carbonate (pH 7.6). Then, 400µl standard solution
was pippeted into a tube to produce a dilution series as shown in Figure 2.3. The diluent
serves as the zero standard (0pg/ml).
Figure 2.3
Serial dilutions for standard curve
Page | 79
2.2.9.2 Human VEGF ELISA
The cells were grown up to ~80% confluence in flasks, detached and counted as
described in section 2.2.1.3. Then, 1x106 cells were seeded to 75cm2 culture flasks and
cultured in growth factor deprived medium (phenol red-free RPMI 1640 medium
containing 10% charcoal stripped FCS) for 48 hours. The conditioned medium (cell
culture supernatant) of each transfected cell lines was collected and 100µl of each
standard (see Figure 2.2) and sample was added into VEGF microplate wells. The plate
was covered with the lid and incubated over night at 4oC with gentle shaking. On the
following day, the conditioned medium was removed completely and the plate was
washed four times with 300µl of wash solution provided by the VEGF Elisa kit. Then,
the biotinylated secondary antibody (100µl) was added to each well and incubated for 1
hour at RT. After removing the unbound second antibody, the plate was washed as
mentioned above and 100µl of HRP-Streptavidin solution was added to the wells and
incubated for 45mins at RT. Discard the solution and repeated the wash step as
mentioned above. Finally, 100µl of TMB substrate reagent was pipetted to each well to
visualize the level of VEGF and incubated for 30mins at RT with gentle shaking
followed by adding 50µl of 2M sulfuric acid to stop the reaction. The plate was then
read at 450nm in the optical density plate reader immediately. The expression level of
VEGF secreted in the conditioned medium for each cell line is quantified against the
standard curve to obtain the concentration of VEGF (pg/ml). The results are the
mean ± SD of three separate experiments.
Page | 80
2.2.10
In vitro angiogenesis assay
In vitro angiogenesis for conditioned medium from each tansfectant was assessed using
the Millipore in vitro angiogenesis assay kit which provided a timesaving system for
evaluation of tube formation by endothelial cells. When cultured on ECMatrix, a solid
gel of basement proteins prepared from the Engelbreth-Holm-Swarm (EHS) mouse
tumor cells, these endothelial cells could achieve fully developed tube structures
between 4-8 hours under the effect of pre-angiogenic factors.
2.2.10.1
Preparation of the ECMatrix coated plate
ECMatrix was thawed on ice overnight prior to the in vitro angiogenesis assay and then
mixed with 10 x dilution buffer in a sterile microfuge tube. The tip of pipette tip was cut
off by a sterile knife to ease pipetting and avoid pipetting air into the solution as
ECMatrix is highly viscous. The diluted ECMatrix (50µl) was transferred to each well
of a 96-well tissue culture plate. All equipments such as pipette tips, plates and tubes
were pre-cooled and all procedures were carried out in a cold room. The ECMatrix
coated plate was incubated at 37oC for 2 hours to allow the matix solution to solidify.
2.2.10.2
Angiogenesis assay
Human umbilical vein cells (HUVEC) were grown to 60-80% confluence in 75cm2
culture flasks in EndoGRO basal medium added with EndoGRO LS supplement kit.
The HUVEC were harvested as described in section 2.2.1.3 and resuspended in
EndoGRO basal medium containing 5% charcoal stripped FCS. Then, 100µl of 1 x 104
HUVEC were seeded onto the surface of the polymerized ECMatrix of each well
followed by adding 100 µl of conditioned medium from each transfected cell line. The
Page | 81
assay plate was incubated at 37oC, 5% CO2 for 6 hours. At the end point of the assay,
cell-tubes were visualized by adding 50µl of crystal violet (0.5% crystal violet in a
solution of 50% ETOH/PBS containing 5% formaldehyde) at RT for 10mins and
quantified using light microscope in 40X magnification.
2.2.10.3
Quantitation of tube formation
Tube formation is a multi-step process, stating with cell migration and alignment,
followed by the development of capillary tubes, sprouting of new capillaries and finally
the formation of the cellular networks. There are several measurements for quantitation
of tube formation such as branch point counting and total capillary tube length
measurement. However, the pattern recognition quantitation method works best in in
vitro angiogenesis assay involving activators of angiogenesis. A numerical value to each
pattern was allocated as presented in Table 2.2. Five random view-fields per well were
examined and the values averaged.
Table 2.2
The degree of angiogenesis progression and its numerical value
Page | 82
2.2.11
General molecular biology methods
2.2.11.1
Calculation of DNA and RNA concentration and purification
The DNA or RNA samples were examined for concentration and quality using the
NanoDrop ND-1000 spectrophotometer. Nucleic acid sample (1µl) was loaded onto the
lower measurement pedestal. After closing the sampling arm, the spectral measurement
was initiated using the operating software on the computer. The DNA or RNA sample
concentration was presented on the screen in ng/µl based on absorbance at 260nm using
the modified Beer-Lambert equation. The purity of the sample was also assessed using
ratio of absorbance at 260nm and 280nm: a ratio of ~1.8 was generally accepted as pure
for DNA; a ratio of ~2.0 was generally accepted as pure for RNA.
2.2.11.2
Ethanol precipitation of DNA and RNA
The DNA or RNA to be precipitated was mixed with 2.5 volumes of chilled state
temperature absolute ethanol and 10% volumes of 3M sodium acetate (pH5.2). The
mixture was mixed gently by inversion and incubated at –80oC freezer overnight for
DNA or RNA to precipitate. The precipitated DNA or RNA was pelleted by centrifuge
at 16,000 rpm at 4oC in a microcentrifuge for 20 mins. The supernatant was removed
and the pellet was washed by 100μl of pre-chilled 70% (v/v) ethanol. The DNA or RNA
was recentrifuged at 16,000rpm at 4oC for 15 mins and the supernatant was decanted.
The DNA or RNA was air dried and resuspended in sterile DEPC-treated water.
Page | 83
2.2.11.3
Agarose gel electrophoresis of DNA
Agarose gel electrophoresis was carried out in 1xTBE buffer (0.089M Tris-base,
0.089M Boric acid, 0.002M EDTA pH8.0). The agarose concentration of the gel was
0.8% to 2.0% (w/v) according to DNA size. The agarose was dissolved in 200ml of
1xTBE buffer by microwave heating for 2mins and cooled to 40-50oC. Then, 10µl of
500ng/ml ethidium bromide was added for visualisation. The gel mixture was poured
and placed at 4oC to solidify. DNA samples were prepared by adding 5x agarose gel
loading buffer [0.25% (w/v) bromophenol blue, 0.25% (w/v) xylene cyanol FF, 30%
(v/v) glycerol in water] to a 1x concentration and loaded into the gel alongside DNA
size marker. The gel was run for 50mins at 90V and visualised on a UV
transilluminator.
2.2.11.4
Purification of DNA from agarose gels
DNA fragments were separated using agarose gel electrophoresis as described
previously in section 2.2.11.3. The required fragments were visualised by ethidium
bromide and excised using a sterile blade. Each excised gel slice was weighed and three
times the volume of buffer QG was added. Then, the mixture was dissolved in a 50oC
water bath for 10 mins with occasional vortex mixing. The melted agarose and DNA
solution was applied to a QIAquick spin column and centrifuged for 1 minute at 16,000
rpm in a microcentrifuge and the flow through was discarded. The membrane, bound
with DNA, was washed by passing through 750l of buffer PE and re-centrifuged for a
further minute to remove any residue. The DNA was eluted from the column by adding
30μl of distilled water and quantified as described above. The DNA was stored at –20oC
until needed.
Page | 84
2.2.11.5
Restriction Enzyme digestion of plasmid DNA
Plasmid DNA was digested with two different restriction enzymes (XhoI and PstI) to
check for the presence of insert or to produce the DNA fragment for ligation. The
restriction enzyme digest mixture was prepared as shown in Table 2.3:
Table 2.3
Water
35µl
10x NEBuffer 3
5µl
XhoI 20units/µl
1µl
PstI 20units/µl
1µl
BSA 10µg/µl
5µl
DNA 1µg/µl
3µl
The restriction enzyme digestion mixture (total volume 50µl)
The restriction enzyme digest mixture was incubated at 37oC for 2 hours to allow
complete digestion of the template DNA. Then, the enzyme reaction was inactivated by
heating to 68oC for 10 minutes and the products were analysed by gel electrophoresis.
Page | 85
2.2.11.6
DNA ligation
Fragments of DNA were removed from pBluescript vector using restriction enzyme
digestion as described in section 2.2.11.5 and inserted into transfection vector
pIRES2-EGFP by ligation reaction using T4 DNA ligase. The ligation mixture was
prepared as shown in Table 2.4.
Insert DNA (180ng/µl)
2µl
Vector DNA ( 110ng/µl)
1µl
2x quick Ligation buffer
10µl
T4 DNA Ligase
1µl
( 400 cohesive end units /µl)
Table 2.4
H2 O
6µl
Total volume
20µl
The ligation reaction mixture
After overnight incubation at 4oC, 5µl of ligation mixture was transformed into
competent bacterial cells as described in section 2.2.12. The recombinant DNA was
recovered from bacterial cells using a Qiagen miniprep or midiprep kit as described in
section 2.2.13.
Page | 86
2.2.11.7
cDNA sequencing
The recombinant plasmid DNAs or cDNA sequences generated by reverse transcription
polymerase chain reaction (RT-PCR) were sent for sequencing at the University of
Liverpool, School of Tropical Medicine. Sequencing was carried out by using a big dye
terminator cycle sequencing reaction kit with an ABI prism 377 automated DNA
sequencer. Sequence alignment was determined by using BioEdit for searching
Genbank (BLAST).
2.2.12
Transformation of competent bacteria with plasmid DNA
2.2.12.1
Preparation of competent bacterial cells
E.coli (DH5) glycerol stock was streaked onto LB-agar plate and incubated at 37C
overnight. On the following day, a single colony was inoculated into a conical flask
containing 10mls LB broth and incubated at 37oC overnight with shaking at 225rpm.
Then, 1ml of overnight culture was transferred into 100ml of SOB medium (2% w/v
bactotryptone, 0.5% w/v yeast extract, 10mM NaCl, and 2.5mM KCl) and incubated at
37C with shaking at 225rpm until the OD550 reached 0.4. The culture solution was
split into 8x 12ml aliquots and placed on ice to cool for 10mins. The DH5 bacteria
were pelletted by centrifuging at 2500rpm for 10mins at 4oC and the supernatant was
discarded. The bacterial cell pellets were resuspended in 8.25ml of pre-cooled RF1
buffer (100mM KCl, 50mM MgCl2.4H20, 30mM KCl, 10mM CaCl2 and 15% v/v
glycerol, pH6.8) and incubated on ice for 10mins. After a further centrifuging at
2500rpm for 10mins at 4oC, the cell pellet was resuspended in 2ml of RF2 buffer
Page | 87
(10mM MOPS, 10mM KCl, 75mM CaCl2.2H2O and 15% v/v glycerol, pH6.8). The
DH5 bacteria solution was dispensed into 1ml aliquots in cryovials, frozen in liquid
nitrogen and transferred immediately to –80oC freezer for storage.
2.2.12.2
Transformation
A vial of DH5 competent cells was removed from the -80 C freezers and thawed on
ice. Then, 50ng of plasmid DNA or 10μl of the ligation mixture was added to 50µl of
competent DH5 cells, mixed by flicking gently and incubated on ice for 30mins. The
cells were heat shocked at 42C for exactly 90 seconds and placed on ice for a further
2mins followed by adding 800l of SOC (2% w/v bactotryptone, 0.5% w/v yeast
extract, 10mM NaCl, 10mM MgCl 2, 10mM MgSO4, 2.5mM KCl, 20mM glucose) and
incubated 37C for 1 hour in a shaking incubator at 225rpm. Transformed bacteria
solution (200l) was plated onto LB plates containing 50µg/ml ampicillin or 30µg/ml
kanamycin for antibiotic selection and incubated at 37C overnight. Finally, colonies
were picked from the plate and grown in LB broth containing antibiotic at 37C,
225rpm overnight and plasmid minipreps were conducted.
Page | 88
2.2.13
Isolation of plasmid DNA
The isolation of plasmid DNA was conducted using extraction kits provided by Qiagen.
2.2.13.1
Miniprep extraction of plasmid DNA
The overnight cultured bacteria (5ml) in LB medium containing antibiotic was
harvested by centrifuging at 6,000rpm for 1min and the supernatant was removed. The
cell pellet was resuspended in 250l of cell suspension buffer P1 (50mM Tris-HCl
pH8.0, 10mM EDTA and 100μg/ml RNaseA) by vortexing, followed by adding 250l
of cell lysis buffer P2 (200mM NaOH and 1% w/v SDS) and mixed by gentle inversion
of the microcentrifuge tube six times. Then, 350l of neutralisation buffer N3 (4.2M
Gu-HCl, 0.9M potassium acetate pH4.8) was added and mixed by gentle inversion of
the microcentrifuge tube six times. The mixture was centrifuged at 13,000rpm for
10mins and the supernatant was loaded to a miniprep spin column. The column was
centrifuged at 13,000rpm for 1min and the flow through was discarded. The column
was then washed by adding 750l of wash buffer PE (10mM Tris-HCl pH7.5 and 80%
ethanol) and centrifuged at 13,000rpm for 1min. After removing the flow through, the
spin column was centrifuged for an additional minute to remove residual wash buffer.
The column was placed in a clean 1.5ml microcentrifuge tube and added with 30l of
distilled water to the centre. After 1min incubation at RT, the plasmid DNA was eluted
by centrifuging at 13,000rpm for 1min. The concentration and quality of the DNA were
examined as described in section 2.2.11.1 and isolated plasmid DNA was stored in a 20C freezer.
Page | 89
2.2.13.2
Midiprep extraction of plasmid DNA
A single colony of transformed DH5 bacteria was transferred into flasks containing
10ml of LB medium with antibiotic and left to grow overnight at 37C, 225rpm. The
overnight cultured bacterial cells (5ml) were then transferred to 200ml LB medium
containing antibiotic and incubated for 5 additional hours at 37C, 225rpm. The
transformed bacterial cells were harvested by centrifuging at 6,000rpm for 10min at 4 oC
and the supernatant was removed. The cell pellet was resuspended in 4ml of buffer P1
(50mM Tris-HCl pH8.0, 10mM EDTA, 100μg/ml RNase A) by vortexing until no
clump was remained. The bacterial cells were lysed by adding 4ml of lysis buffer P2
(200mM NaOH, 1% w/v SDS), gently mixed by six times inversion. After incubation at
RT for 5mins, 4ml of pre-chilled P3 neutralising buffer (3.0M potassium acetate pH5.5)
was added and the mixture was mixed by inverting six times, incubated on ice for
15mins and then centrifuged at 16,000rpm, 11C for 30mins. The supernatant
containing plasmid DNA was transferred to a clean 30ml centrifuge tube and recentrifuged at 16,000rpm, 11C for further 15mins. In the mean while, a Qiagen
midiprep column was equilibrated by applying 4ml of equilibration buffer QBT
(750mM NaCl, 50mM MOPS pH 7.0 and 15% v/v isopropanol) and allowed to empty
by gravity flow. Once the Qiagen midiprep column was equilibrated, the cleared
supernatant containing plasmid DNA was loaded into the column and allowed to flow
under gravity. The column was washed twice with 10ml of wash buffer QC (1.0M
NaCl, 50mM MOPS pH 7.0, 15% v/v isopropanol) and the plasmid DNA was eluted
from the column using 5ml of elution buffer QF (1.25 M NaCl, 50mM Tris-HCl pH 8.5,
15% v/v isopropanol). The eluted DNA was precipitated by adding 3.5ml of
isopropanol, mixed and centrifuged at 4C, 11,000rpm for 30mins. The supernatant was
Page | 90
carefully removed and the pellet was washed with 2ml of 70% (v/v) ethanol and
centrifuged at RT, 11,000rpm for 10mins. After removing the supernatant, the pellet
was air-dried for 10mins and the plasmid DNA was then dissolved in 200l of nucleasefree water. The concentration and quality of the DNA were examined as described in
section 2.2.11.1 and the isolated plasmid DNA was stored at -20C
2.2.14
Mutagenesis of C-FABP gene and construction of CFABPs transfection vectors
In previous works, a 436 base pair fragment of the protein coding region of C-FABP
had been amplified by RT-PCR from total mRNA isolated from PC-3M cells. The
amplified C-FABP product was T-A cloned into a PGEM-Teasy plasmid and then
transferred into pBluescript II SK vector as shown in Figure 2.4. Transformertm SiteDirected Mutagenesis Kit was used to generate one or two point mutations to the CFABP gene. The transformer mutagenesis strategy, as shown in Figure 2.5, can not only
introduce single or multiple specific base mutations with high efficiencies (70-90%) but
also benefit by avoiding subcloning and using single-stranded vectors or specialized
double-stranded plasmids. Furthermore, the use of T4 DNA polymerase instead of PCR
reduces the risk of generating spurious mutations.
 Firstly, the mutagenic primer and the selection primer containing a mutation in a
restriction enzyme site are annealed to one strand of the denatured doublestranded plasmid.
Page | 91
 Secondly, after DNA extension, ligation and a primary selection by restriction
digest the mixture of mutated and unmutated plasmids is transformed into a mutS
E.coli strain defective in mismatch repair.
 Thirdly, the DNA was isolated from pooled transformants and digested with the
selective restriction enzyme.
 Finally, the selective digested DNA was transformed into DH5α bacterial cells
and the mutated plasmid was recovered by midiprep extraction.
Page | 92
Figure 2.4
Circular maps and lists of features for the pBluescript II SK
vector
The pBluescript II SK vector contains an ampicillin selection
marker which is essential for Transformer site-directed
mutagenesis kit because the bacterial strain BMH 71-18 mutS has
a Tn10 transposon and therefor is tetracycline resistant. The
efficiency of the chosen restriction enzyme digestion has been
examined at a low DNA concentration (0.1µg/30µl) before the
mutagenesis assay procedure.
Page | 93
Figure 2.5
Transformer site-directed mutagenesis strategy (The picture is taken
from Clontech transformer site-directed mutagenesis kit user manual)
Page | 94
2.2.14.1
Selection and mutagenic primers
Selection primer:
5’- CAAGATCTCGATGGCGGTGGCG-3’
Mutagenic primer (R109A): 5’- CGTGTTATGTCGTTTTAACTTTC -3’
Mutagenic primer (R129A): 5’- GTGGACATGACGCTAGATAC -3’
The changed base pairs of the selection and mutagenic primers are underlined. All
primers were synthesized with an incorporated 5’ phosphate.
2.2.14.2
Generation of the mutant plasmid DNA
The primer/plasmid annealing reaction mixture was prepared with 2µl of 10x annealing
buffer (200mM Tris-HCl pH7.5, 100mM MgCl2, 500mM NaCl), 0.1µg plasmid DNA,
2µl of 0.1µg/µl selection primer, 2µl of 0.1µg/µl mutagenic primer (the selection and
mutagenic primers are listed in section 2.2.14.1) and nuclease-free water (adjust to a
total volume of 20µl). After denaturation at 100oC for 3mins, the reaction mixture was
immediately placed on ice for 5mins. Then, the reaction mixture was added with 3µl of
10x synthesis buffer, 1µl of T4 DNA polymerase (3units/µl), 1µl of T4 DNA ligase
(2.84 WEISS units/µl), 5µl of nuclease-free water and incubated at 37oC for 2 hours to
synthesize the mutant DNA strand. The reaction was stopped by heating at 70oC for
8mins and cool down to RT.
2.2.14.3
Selection by restriction digestion
The DNA in synthesis/ligation mixture was ethanol precipitated as mentioned in section
2.2.11.2 and resuspended in restriction enzyme digestion mixture with 0.5µl of NotI
(20units/µl), .3µl of 10x NEBuffer3 and 26.5µl of nuclease-free water for primary
selection. The reaction mixture was incubated at 37oC for 2 hours and heated at 70oC for
Page | 95
5mins to stop the digestion. Then, 11µl of the digested plasmid DNA was transformed
to BMH 71-18 mutS competent cells as described in section 2.2.12. The mixed plasmid
pool was isolated using miniprep as described in section 2.2.13.1 and digested with NotI
restriction enzyme. The digested plasmid DNA was transformed to DH5α competent
cells followed the same procedure as for the first transformation. The mutant DNA was
recovered by miniprep or midiprep and the mutation was verified by directly sequencing
the mutagenized region as previously described in section 2.2.11.7. The mutagenic
primer (R109A) was applied to introduce single mutation and the double mutations
were generated using both mutagenic primer (R109A) and mutagenic primer (R129A).
2.2.15
Total RNA isolation
Total RNA was isolated from cultured cells using the RNAeasy Mini Kit. Two 75cm2
flasks cells were cultured to 60-80% confluence and harvested as described in section
2.2.1.3. The cells were washed with PBS which was removed by centrifuging at RT,
900rpm for 3mins and 350µl buffer RLT containing 3.5µl -Mercaptoethanol was
added to lyse the cells. The cell lysate was then homogenised by centrifuging through a
PrepEase filter unit at 16,000rpm for 1min and precipitated in one volume of 70%
ethanol. The resulting solution was transferred to an RNAeasy mini column sitting in a
2ml collection tube and centrifuge for 30sec at 11,000rpm. The column was then
washed by 700μl buffers RW1 and twice with 500μl RPE buffer by centrifuging at
11,000rpm for 30sec. The RNeasy spin column was centrifuged again at 16,000rpm for
1min to eliminate any carryover buffer. After 30μl of RNase free water was added, the
RNeasy spin column was incubated at RT for 1min. Finally, the total RNA was eluted
Page | 96
by centrifuging the column for 1min at 16,000rpm. The total RNA yield and purity was
determined by NanoDrop ND-1000 spectrophotometer as mentioned in section 2.2.11.1.
2.2.16
Reverse transcription polymerase chain reaction
2.2.16.1
First strand cDNA synthesis
First strand cDNA was synthesized using total RNA isolated from LNCaP cells as
described in section 2.2.15. Total RNA (3μg) was mixed with 1μl of 50μM Oligo (dT)20
primer and 2μl of dNTP mixture (10mM each dATP, dCTP, dGTP, dTTP at neutral
pH). After the volume was adjusted to 13μl with nuclease-free water, the mixed solution
was incubated at 65°C for 5mins and chilled on ice for 1min. Then, the solution was
mixed with 4μl of 5x first strand buffer, 1μl of 0.1M DTT, 1μl of RNaseOUT
(40units/μl) and 1μl of SuperScriptIII reverse transcriptase (200units/μl). The reaction
mixture was incubated at 50oC for one hour, followed by incubation at 70oC for 15mins
to inactivate the reaction. The First strand cDNA was used as a template for
amplification.
Page | 97
2.2.16.2
Polymerase chain reaction
Polymerase chain reaction (PCR) was performed to amplify a specific region of the
DNA template. The forward and reverse primers are listed as below:
Forward primers sequence:
5’- ACCATGGCCACAGTTCAGCA -3’
Reverse primers sequence:
5’- CCTGTCCAAAGTGATGATGGAA -3’
The DNA template was amplified in a total volume of 50μl reaction mixture as shown
in table 2.5.
DNA template
50-200ng
10x PCR buffer
5µl
MgCl2 (25mM)
3µl
Forward primer (10µM)
1µl
Reverse primer (10µM)
1µl
dNTPs mixture
(10mM each)
Taq polymerase
( 5U /µl)
Nuclease-free water
Table 2.5
1µl
1µl
Up to 50µl
PCR mixture
The reaction was incubated in a thermal cycler at 95oC for 5mins, followed by 35 cycles
of PCR amplification, each cycle consisting of denaturing: 95oC for 30 seconds;
annealing: 55oC for 45 seconds; extension: 72oC for 1 min, followed by one cycle
incubation at 72oC for 5mins and then maintained the reaction at 4oC. PCR
amplification products were analysed by agarose gel electrophoresis and purified as
previously described in section 2.2.11.3 and 2.2.11.4.
Page | 98
2.2.17
Real-time PCR
The real-time PCR, also known as quantitative real time polymerase chain reaction
(qPCR), was applied in order to quantify the level of C-FABP mRNA in the transfected
LNCaP cells. Traditional PCR or RT-PCR uses agarose gel electrophoresis for detection
of amplified DNA fragments at the end of the reaction. However, real-time PCR is
designed to collect data in the exponential growth phase when the reaction is in
progress, which significantly increased the accuracy for DNA and RNA quantitation
compare to the traditional PCR or RT-PCR.
Several methods exist for real-time quantitation of amplification products, including
fluorescence resonance energy transfer techniques using fluorescently labeled molecular
beacons, SYBR Green I. During the PCR amplification, the fluorescence of SYBR
Green I increases 100- to 200-fold when bound to double stranded DNA as presented in
Figure 2.6 The number of amplicons generated is directly proportional to the increase in
reporter fluorescent signal which was detected at the end of the elongation step of the
PCR reaction by Chromo4 fluorescence detector.
Figure 2.6
SYBR Green Dye binds to the double stranded DNA and fluoresces
Page | 99
2.2.17.1
Real-time PCR primer design
The real-time PCR primers were designed spanning exon-exon junctions of C-FABP
gene in order to avoid amplifying any genomic DNA contamination. The size of PCR
amplicon was limited to between 50bp to 200bp and the primers self complimentarity
and hair pins was also checked as SYBR Green Dye bound to all double stranded PCR
products, including non-specific PCR products. Similarly, the house-keeping gene, β
actin primers were designed as listed below:
Real-time PCR primers for C-FABP
Forward primers sequence:
5’- CATTGGTTCAGCATCAGGAG -3’
Reverse primers sequence:
5’- TTCATGACACACTCCACCACT -3’
Real-time PCR primers for β actin
Forward primers sequence:
5’- ACCATGGCCACAGTTCAGCA -3’
Reverse primers sequence:
5’- CCTGTCCAAAGTGATGATGGAA – 3’
2.2.17.2
Relative real-time PCR
The total RNAs were extracted from each cell line using RNAeasy Mini Kit as
described in section 2.2.15 and reverse transcribe 1µg of total RNA to cDNA using
SuperScriptIII reverse transcriptase as mentioned in section 2.2.16.1. The real-time PCR
mixture for both C-FABP and β actin was prepared with 5µl of 2× Brilliant SYBR
Green qPCR master mix (containing SureStart Taq DNA polymerase, dNTPs mixture,
MgCl2 and optimized buffer), 1µl of forward primer, 1µl of reverse primer, 1µl of
cDNA generated by reverse transcription and 2µl of nuclease-free water. After gentle
Page | 100
mix, the reactions were centrifuged briefly and placed to a real-time PCR thermocycler.
The real-time PCR program is listed in table 2.6.
Temperature
Time
Step1 Denaturation
95oC
15mins
Step2 Denaturation
94oC
15 seconds
Step3 Annealing
60oC
30 seconds
Step4 Extension
72oC
30 seconds
Step5 Plate reading
57oC
15 seconds
Step6 Go back to step 2 and repeat 38 cycles
Step7 Final extension
72oC
10mins
Step8 Melting curve
65-95oC
1oC increment for
10mins
Table 2.6
Real-time PCR program for amplification of short target DNAs (50-400bp)
2.2.17.3
Relative quantitation analysis
Melting curve analysis was carried out to detect the presence of nonspecific products
and the primer dimmers. The relative fold differences of C-FABP mRNA between
transfected cell lines and parental cell line LNCaP were obtained using the formula
listed below:
Relative fold difference=2 -ΔΔCt

ΔCt was calculated as the average Ct for the gene of interest minus the average Ct for the house
keeping gene, β actin.

ΔΔCt was calculated as the ΔCt of the test sample minus the ΔCt of the calibrator sample
Page | 101
2.2.18
Analysis of protein expression using Western blot
2.2.18.1
Isolation of protein extracts from cultured cells
Cells were selected for protein isolation when they were grown to approximately 80%
confluence. The cells were detached (As described in section 2.2.1.3) and suspended in
10ml of completed medium to inactive the trypsin. Cells suspension was transferred to a
25 ml sterile universal tube, centrifuged at 900 rpm for 5 mins, decanted the supernatant
and washed with PBS which was removed by centrifuging. The cells was lysed by
adding CelLytic-M reagent and incubated on a roller mixer for 15 mins at RT (5x106
cells / 100µl). The mixture was transferred into a 1.5ml microcentrifuge tube and then
centrifuged for 20 mins at 10,000rpm to pellet the cellular debris. The supernatant was
harvested into a fresh microcentrifuge tube and the pellet was discarded.
2.2.18.2
Determination of protein concentration
The concentration of protein extract was quantified using the Bradford dye-binding
assay. The dye reagent was diluted 5-fold by adding distilled water (Bradford
reagent: H2O = 1:4) and filtered through Whatman 540 paper. A concentration standard
curve was created by measuring the absorbance (595nm) of a serial dilution of BSA
standards (from 50µg/µl to 500µg/µl) after 15 mins incubation with Bradford reagent.
Protein samples were also diluted by PBS and incubation with Bradford reagent for 15
mins before measuring the absorbance at 595nm. The sample concentration was
calculated using the equation from established standard curve.
Page | 102
2.2.18.3
Sodium dodecyl sulphate polyacrylamide protein gel electrophoresis
(SDS-PAGE)
Proteins isolated from cells were analysed using the Bio-Rad miniprotean system with 1
mm spacers. Proteins were separated by SDS-PAGE with 4% (w/v) acrylamide stacking
gel and 10% or 15% (w/v) acrylamide resolving gel according to the molecular size of
the target protein. The mixture (20μl) of 2x loading buffer containing equal amount of
protein (v/v) was heated at 100oC in a water bath for 10 mins prior to loading, which
linearized the protein by breaking hydrogen bonds of the tertiary structure of the
polypeptide and reveal the antigen to be recognised by the antibody. The mixture was
then chilled on ice for 2 mins and spun down before loading into the SDS gel.
Electrophoresis was performed in 500ml of 1x running buffer (50 mM Tris-HCl, 200
mM glycine and 0.1% SDS) at 80V through the stacking gel and 160V through the
resolving gel until the dye front had reached the end of the resolving gel.
2.2.18.4
Transfer of proteins from SDS gel to nitrocellulose membrane
Separated Proteins were transferred from SDS gel to a nitrocellulose membrane using
the BioRad mini trans-blot system. Six sheets of Whatman 3mm filter paper and the
nitrocellulose membrane were cut according to the size of the SDS gel and soaked in 1x
transfer buffer for 5 mins. The cassette, with black side down, was placed on a tray
containing transfer buffer and assembles the cassette in following order (from black
side): a pre-wet fiber pad, three sheets of Whatman filter paper, equilibrated SDS gel,
nitrocellulose membrane, three sheets of Whatman filter paper and another pre-wet fiber
pad. The air bubbles were removed from each step using a glass roller. The transfer was
Page | 103
performed at 100V, 4oC for 1.5 hours in pre-chilled 1x transfer buffer. At the end of the
process, the transfer efficiency was assessed through the Coomassie blue staining: the
gel was stained with Coomassie blue for an hour at room temperature before being
destained in destaining buffer overnight.
2.2.18.5
Immunoblotting for detection of protein expression
The nitrocellulose membrane containing the proteins was blocked with 10ml of 5%
Protoblock for an hour at room temperature on a gentle agitation to prevent non-specific
binding of the primary antibody. The membrane was then incubated on a shaker with a
primary antibody in an appropriate concentration (Table 2.7) at 4 oC overnight. The
following day, the membrane was washed with 1x T-TBS 4 times for 10 mins each time
to remove unbound primary antibody and incubated with a secondary antibody in an
appropriate concentration (Table 2.7) for 2 hours at room temperature. Then, the
membrane was washed with 1x T-TBS 4 times for 10 mins each time again. The probed
antigens on the membrane were visualized by incubation in ECL reagents for 2 minutes
at room temperature. Chemiluminescent images were recorded on Kodak films with
0.5-10 mins exposure. The films were developed and fixed in the dark room.
Page | 104
Target protein
C-FABP
Primary antibody
Monoclonal Rabbit Anti-human
C-FABP (1:200)
VEGF
Polyclonal Rabbit Anti-human
VEGF (1:500)
β-Actin
Monoclonal Mouse
Anti-β-Actin (1:5000)
Table 2.7
Secondary antibody
Polyclonal Swine Anti-rabbit
Immunoglobulins/HRP
(1:1000)
Polyclonal Swine Anti-rabbit
Immunoglobulins/HRP
(1:1000)
Polyclonal Rabbit Anti-mouse
Immunoglobulins/HRP
(1:2000)
Primary and secondary antibodies used in Western blot
To standardize the loading difference, the level of β-actin expression was examined.
The expression level (EL) of each target protein was calibrated using the formula listed
below:
Normalised EL of target protein = EL of target protein / EL of β-Actin
Page | 105
2.2.19
Statistic method
The Student’s t-test, introduced in 1908 by William Sealy Gossett, is the most
commonly used method to evaluate the differences in averages between two groups. In
this thesis, the Student’s t-test was used to compare any differences observed between
each experimental group and the control group. The results from most of assays were
statistically assessed using the Student’s t-test. For all Students’s t-test calculated by
Microsoft Office Excel 2003, the P-value less than 0.05 was regarded as statistical
significance.
Page | 106
Chapter 3 Transfection of wild type and mutant
C-FABP into LNCaP prostate cancer cells
3.1
Introduction
As previously mentioned in Chapter 1, cutaneous fatty acid binding protein (C-FABP)
is a FABP family member binding to long chain fatty acids with high affinity. C-FABP
has been identified as a gene which was not only involved in malignant progression of
prostate cancer but also able to promote the growth of primary tumors and induce
metastasis when it transfected into benign rat Rama 37 model cells [157]. Recently, it
was demonstrated that C-FABP was a prognostic marker for patient outcome and
a target of tumor-suppression for prostate cancer [159].
Previous studies suggested that C-FABP might promote the malignant progression of
prostate cancer by up-regulating the expression of the gene for vascular endothelial
growth factor (VEGF), a potent factor for angiogenesis which is essential for growth
and expansion of solid tumours [158, 159, 164]. However, how C-FABP up-regulates
the expression of VEGF is still not clear. Since the common biological function of
FABPs, including C-FABP, is to transport the intracellular fatty acids into cells,
therefore it is interesting to know whether the tumourigenicity-promoting function of
C-FABP is related to its activity of transporting fatty acids. It has been confirmed that
there are three key amino acids (Arg109, Arg129, and Tyr131) of C-FABP, which are
highly conserved amongst the FABP protein family, and which are responsible for
Page | 107
binding to the carboxylate group in the fatty acids [165]. Mutation of one or two of the
three key amino acids would either partially or completely deprive of C-FABP fatty
acid-binding ability.
In order to investigate whether binding to fatty acids is essential for C-FABP to promote
cancer malignant progression, one and two site-directed point mutations were
introduced to the C-FABP cDNA region which contains the fatty acid-binding motif.
The mammalian expression vector pIRES2-EGFP containing wild type and mutated
C-FABP cDNAs were tansfected respectively into the LNCaP prostate cancer cells,
which did not express C-FABP prior to the transfection, to establish cell lines
expressing wild type C-FABP and mutated C-FABPs.
Page | 108
3.2
Results
3.2.1 Generation of mutations in C-FABP cDNA
Point mutations were introduced into fatty acid binding domain of the C-FABP cDNA
using Transformertm Site-Directed Mutagenesis Kit as described in Chapter 2 section
2.2.14. The pBluescript II SK vectors containing wild type, single-mutation and
double-mutated C-FABP cDNA were transformed into DH5α E.coli competent cells
respectively for amplification. After overnight incubation at 37oC, 225rpm, the plasmid
vectors were isolated from DH5α cells using midiprep and the presence of mutations
were confirmed by nucleotide sequence analysis using specific C-FABP cDNA forward
and reverse primers as described in Chapter 2 section 2.2.16.2. The constructed
harboring wild type of C-FABP cDNA was designated as C-FABP-WT. Those
containing the single and double-mutated C-FABP cDNAs were denoted as C-FABP109A and C-FABP-109/129A, respectively.
As showing in Figure 3.1, sequencing analysis results demonstrated that the sequence of
the C-FABP cDNA region containing the first mutation site was successfully altered by
converting Arginine 109 to Alanine 109. Thus the triplet AGA was changed into GCA.
In the second mutation site the triplet CGG was changed into GCG by replacing
Arginine 129 with Alanine 129. In the construct of C-FABP-109A, only one amino acid
was changed in first mutation site and no change was made in second mutation site. In
the construct of C-FABP-109/129A, amino acids in both mutation sites were changed.
Page | 109
Figure 3.1
Detection of mutations in C-FABP cDNA by sequencing analysis
(A) Point mutantion in first site is shown in panel A. The Alanine
(GCA), mutated from Arginine (AGA), is presented both in
C-FABP-109A and C-FABP-109/129A constructs. (B) Point mutation in
second site is shown in panel B. The Alanine (GCG), converted from
Arginine (CGG), is presented in C-FABP-109/129A but not in
single-mutated C-FABP, C-FABP-109A and C-FABP-WT constructs.
Page | 110
3.2.2 Insertion of wild type C-FABP and mutant C-FABPs into a
mammalian expression vector - pIRES2-EGFP
The C-FABP-WT, C-FABP-109A and C-FABP-109/129A were removed from
pBluescript II SK vector by restriction enzyme digestion using XhoI/PstI
restriction enzymes. The products of restriction enzyme digestion were separated
by agarose gel electrophoresis (1%) as presented in Figure 3.2A. The DNA fragments
of C-FABP-WT, C-FABP-109A and C-FABP-109/129A were recovered from agarose
gel respectively as described in Chapter 2 section 2.2.11.4. The mammalian expression
vector pIRES2-EGFP was linearized using XhoI/PstI restriction enzymes as shown in
Figure 3.3A and purified from agarose gel. The purified DNA fragment of wild type and
mutant C-FABPs were inserted into pIRES2-EGFP vector respectively using T4 DNA
ligase. The recombinant DNAs were transformed into DH5α bacterial cells and plated
onto LB plates in the presence of antibiotics (50µg/ml ampicillin). Single colonies of
transformed DH5 bacterial was transferred into flasks containing 10ml of LB medium
with 50µg/ml ampicillin and left to grow overnight at 37C, 225rpm. The overnight
cultured bacterial cells (5ml) were harvested and the pIRES2-EGFP vectors containing
C-FABP-WT, C-FABP-109A and C-FABP-109/129A were isolated from bacterial cells
respectively using miniprep as described in Chapter 2 section 2.2.13.1. The ligation was
examined using restriction enzyme digestion as shown in Figure 3.3B. The sequencing
analysis also confirmed the presence of the insertions in pIRES2-EGFP vector as shown
in Appendix.
Page | 111
A
B
1
Figure 3.2
2
3
4
M
The products of restriction enzyme digestion for the ligation reaction
(A) After DNA sequencing analysis, the pBluescript II SK vector
containing C-FABP-WT (Lane 2), C-FABP-109A (Lane 3) and CFABP-109/129A (Lane 4) was digested by XhoI/PstI for the ligation
reaction. The control vector digested by XhoI/PstI is presented in lane1.
For lanes 2, 3 and 4, the bottom band represents DNA fragment of CFABP-WT, C-FABP-109A and C-FABP-109/129A respectively. The top
band in lanes 1, 2, 3, and 4 represents digested pBluescript II SK vector
(3.0 kb). (B) The DNA standard marker is denoted in number of base
pairs.
Page | 112
A
B
1
Figure 3.3
2
M
1
2
3
4
5
M
Identification of the recombinant DNAs by restriction enzyme digestion
(A) Two restriction enzyme digestions (Lanes 1 and 2) of mammalian
expression vector pIRES2-EGFP (5.3 kb) using XhoI/PstI restriction
enzymes to linearize the plasmid for the ligation reaction. (B) After
ligation reaction, the recombinant DNAs were digested using
XhoI /BamHI to assure that the DNA fragment of C-FABP-WT,
C-FABP-109A and C-FABP-109/129A was inserted into the vector. The
empty pIRES2-EGFP vector was used as control in lane 1. The vector
containing C-FABP-WT digested with BamHI was shown in lane 5 for
comparison with the recombinant DNAs digested with XhoI/PstI
restriction enzymes. For lanes 2, 3 and 4, the bottom band represents
DNA fragment of C-FABP-WT, C-FABP-109A and C-FABP-109/129A
respectively. The top band in lanes 1, 2, 3, 4, and 5 represents digested
pIRES2-EGFP vector. In panel A and B, the DNA standard marker in
lane M is denoted in number of base pairs.
Page | 113
3.2.3 Detection of wild type and mutated C-FABP mRNAs in
transfected LNCaP cells by RT-PCR
LNCaP cells were stably transfected with the empty pIRES2-EGFP vector and the
vector containing C-FABP-WT or one of the two mutated C-FABP: C-FABP-109A and
C-FABP-109/129A using a polyamine based transfection reagent. The cell line
transfected with C-FABP-WT, C-FABP-109A, C-FABP-109/129A and the empty
pIRES2-EGFP vector were designated LNCaP-WT, LNCaP-R109A, LNCaPR109/129A and LNCaP-V.
It was difficult to establish transfected clones as LNCaP cells had an extremely low
survival rate when plated at very low concentrations in tissue culture flask [166].
Furthermore, the Geneticin was also significantly slow the growth rate of the transfected
cells in selection step which makes the formation of transfected clones more difficult. In
order to overcome the difficulties in generating LNCaP clones, the modified
transfection method was developed using GeneJammer transfection reagent which is a
proprietary formulation of polyamine offering high efficiency with low cytotoxity. After
selection step, the survived clones were analyzed by fluorescence microscopy as
described in Chapter 2 section 2.2.2.2.
The successful transfected clones for LNCaP-WT, LNCaP-R109A, LNCaP-R109/129A
and LNCaP-V were isolated using ring clone and cultured in selective medium
separately. The pool of wild type C-FABP and mutant C-FABP transfected clones was
Page | 114
also established by random selection of five single clones. The abundant green
fluorescence images for pooled transfected cell lines were presented in Figure 3.4
confirmed the expression of recombinant pIRES2-EGFP vectors. For LNCaP-WT,
LNCaP-R109/129A and LNCaP-V transfected cell lines, the fluorescence-positive cells
represented a subset of approximately 90% of the whole population. For LNCaP-R109A,
approximately 80% of cells displayed strong green fluorescent emission and about 10%
of LNCaP-R109A transfectant expressed less green fluorescence. The rest 10%
populations of all four pooled transfected cell lines were fluorescence-negative. The
fluorescence-positive cell population stayed at same level when increased the
concentration of Geneticin or removed the antibiotic for a period of time (4 weeks).
Similar observations were found in single cloned transfectants.
Page | 115
Figure 3.4
The green fluorescent emission images of successful transfected cells
The green fluorescence images of pooled LNCaP-WT, LNCaP-R109A,
LNCaP-R109/129A and LNCaP-V were shown in panel A1, A2, A3 and
A4 respectively. All four cell lines appeared strong green fluorescence
confirmed the expression of recombinant pIRES2-EGFP vectors.
Page | 116
To confirm the presence of the wild type and mutated C-FABP mRNAs existence in the
transfected cell lines, total RNA was isolated from each pooled clones of transfectants.
The total RNAs were amplified by PCR using a specific C-FABP forward primer which
was listed in Chapter 2 section 2.2.16.2 and a specific pIRES2-EGFP plasmid reverse
primer to ensure the amplification products were from the transgenes instead of the
endogenous gene for C-FABP. The products of PCR amplification were analysed by
agarose gel electrophoresis as shown in Figure 3.5. No band was detected in PCR
amplification without reverse transcriptase indicating that the amplified bands were not
arising from total RNA isolation. The RT-PCR products on the agarose gel were excised
and purified as previously mentioned in Charpter 2 section 2.2.11.4 and send for
sequencing analysis (Liverpool University, School of tropical Medicine, Sequencing
Unit).
The RT-PCR products of LNCaP-R109A and LNCaP-R109/129A were the same size as
LNCaP-WT (623kb). In contrast, no band was detected in LNCaP-V and negative
control amplifications. Subsequent nucleotide sequencing also confirmed the presence
of C-FABP-WT, C-FABP-109A, and C-FABP-109/129A.
Page | 117
Figure 3.5
Detection of mRNAs from transfected cell lines by RT-PCR
Reverse transcriptase PCR was carried out using the Taq polymerase
with specific primers to amplify the wild type and mutated C-FABP
gene. Total RNAs were isolated from LNCaP cell line (Lane 1) and
transfected cell lines LNCaP-V (Lane 2), LNCaP-WT (Lanes 3 and 4),
LNCaP-R109A (Lanes 5 and 6) and LNCaP-R109/129A (Lane 7 and 8).
Negative controls are presented in lanes 4, 6 and 8 in which the reverse
transcriptase was omitted. The PCR products were analyzed on a 2%
agarose/EtBr gel in 1xTBE buffer alongside 100 base pair ladder DNA
marker (Lane M).
Page | 118
3.2.4 Expression of wild type and mutant C-FABP proteins in
LNCaP cell line
Levels of wild type and mutant C-FABP proteins expressed in transfected LNCaP cells
were examined by Western Blot as described in Chapter 2 section 2.2.18. The LNCaP-V
cells and highly malignant prostate cancer cells, PC-3M were used as negative and
positive control respectively. The C-FABP specific primary antibody used in Western
Blot analysis was provided by our Japanese colleagues. The level of β-actin expression
in each cell line was also examined and used as loading control. The C-FABP
expression in transfectant cells was quantified by relating to that in positive control cells
using scanning densitometry.
The expression of wild type C-FABP (15 KDa) in LNCaP-WT and PC-3M cells are
shown in Fegure 3.6A and B. When the level of C-FABP expression in positive control
PC-3M cells was set as 1, the level of C-FABP in LNCaP-WT was significantly
increased (Students t-test P<0.05) to approximately 70% of that expressed in the highly
malignant PC-3M cells. No band was observed in LNCaP-R109A, LNCaP-R109/129A
and LNCaP-V. In addition, no significant difference was detacted in wild type C-FABP
levels between single cloned and pooled LNCaP-WT cells (Students t-test P>0.05). The
expression of β-actin, served as a loading control, is also examined as shown in Fegure
3.6A at the size of 42 KDa.
Page | 119
A
B
Figure 3.6
Detection of protein expression levels of wild type C-FABP in single
cloned and pooled transfectant LNCaP cell lines
(A) Cellular lysates from three individual cloned (Lanes 3, 5 and 7) and
three pooled (Lane 4, 6 and 8) of transfectant lines: LNCaP-WT
(Lanes 3 and 4), LNCaP-R109A (Lanes 5 and 6) and LNCaP-109/129A
(Lanes 7 and 8), were subjected to Western blot analysis. C-FABP
protein band was recognized by a monoclonal anti-human C-FABP
antibody and visualized as described in Chapter 2 section 2.2.18. The
β-actin antibody was used to correct the possible loading artifacts. (B)
The relative levels of C-FABP expression in different transfectants. The
results (mean±S.D. of three experiments) were obtained by densitometry
analysis of the band intensities.
Page | 120
Both wild type and mutant C-FABPs were picked up using a rabbit polyclonal antihuman C-FABP antibody. It was observed that intensities of the bands in LNCaP-WT,
LNCaP-109A and LNCaP-109/129A were very similar, whereas the expression of
C-FABP in control cell line LNCaP-V and parental cell line LNCaP is barely detectable
as shown in Figure 3.7A. The β-actin protein expression level in each cell lines, served
as a loading control, was also presented. Further relative quantifications by scanning
densitometry were shown in Figure 3.7B. When wild type C-FABP expression in
LNCaP-WT was set as 1, the level of mutant C-FABPs were 1.09 and 0.946 in
LNCaP-R109A and LNCaP-109/129A cells respectively. No significant expression
differences have been found between the level of wild type C-FABP and level of mutant
C-FABPs (Students t-test P>0.05) in the transfectants. LNCaP-V with empty
pIRES2-EGFP vector and parental LNCaP cells exhibited undetectable level of
C-FABP.
Page | 121
A
B
Figure 3.7
Detection of protein expression levels of wild type and mutant
C-FABPs in pooled transfected LNCaP cell lines
(A) The mutant C-FABP proteins in LNCaP-R109A cells (Lane 2) and
LNCaP-109/129A cells (Lane 3) were recognized using a polyclonal
anti-human C-FABP antibody. The wild type C-FABP in LNCaP-WT
cells (Lane 1) was also detected by the antibody. LNCaP-V and LNCaP
cells were used as negative control in Lane 4 and 5 respectively. (B) The
mean and S.D. of the relative band intensities of each cloned and pooled
cell lines from three individual experiments.
Page | 122
3.2.5 Measurement of levels of wild type and mutant C-FABP mRNAs
in different transfectants by Real-time PCR
To confirm that the transfected wild type and different mutant cDNAs were expressed,
the relative C-FABP mRNA levels in each transfectant cell line was measured by
real-time PCR. The parental cells, LNCaP were used as the calibrator. Total RNAs from
each cell lines were isolated routinely and purified by RNAeasy Mini column.
As poor quality RNA samples can lead to spurious Real-time PCR results, the integrity
and quality of the total RNAs were determined by Agilent 2100 bioanalyzer using RNA
6000 Nano Kit. The results (as shown in Figure 3.8A) were analyzed by the 2100
Expert Software and visual output were confirmed that the isolated total RNAs were in
high quality with both rRNA bands (18S/28S) distinguishable as shown in Figure 3.8B.
The RNA integrity numbers (RIN) for each isolated RNA were between 9.0 to 9.6 out
of 10 and no degradation was observed. The RNA concentration of each sample was
also calculated by the 2100 Expert Software.
After RNA purification, relative quantification real-time PCR was preformed to
measure the mRNA levels of wild type and mutant C-FABPs using exon-exon junction
primers as described in Chapter 2 section 2.2.17. Figure 3.9 summarizes the relative
level of C-FABP mRNA in total RNA isolated from each pooled cell line. The
LNCaP-WT, LNCaP-R109A and LNCaP-R109/129A cell line showed a significant
increase in the relative C-FABP mRNA level when compared to parental cell line
Page | 123
LNCaP. When the C-FABP mRNA level in the parental cell line LNCaP was set at 1,
the levels of expression was 137.5±18.1, 114.1±15.6, and 115±25.2 in LNCaP-WT,
LNCaP-R109A and LNCaP-R109/129A, respectively. On the other hand, the C-FABP
mRNA level in LNCaP-V cells was only slightly increased. The C-FABP mRNA level
between LNCaP-R109A and LNCaP-R109/129A did not show significant difference
(Students t-test P>0.05). Similarly, no difference was found when they compared to
LNCaP-WT (Students t-test P>0.05). However, the relative levels of C-FABP mRNA
expressed in
all
three transfectant lines
(LNCaP-WT,
LNCaP-R109A and
LNCaP-R109/129A) were significantly increased when compared to the control
LNCaP-V cells (Students t-test P<0.05).
Page | 124
A
B
Figure 3.8
Quality checks of total RNA samples by Agilent 2100 bioanalyzer
The quality and quantification of total RNA were assessed using the
RNA 6000 Nano LabChip on Agilent 2100 bioanalyzer. The gel
electropherogram images for LNCaP-WT (Lane 1), LNCaP-R109A
(Lane 2), LNCaP-R109/129A (Lane 3), LNCaP-V (Lane 4) and LNCaP
(Lane 5) were shown in panel A. The fluorescence plots with two peaks
of 18S and 28S ribosomal RNAs of each sample were shown in panel B.
The qualities of RNAs were presented by RINs which were calculated
from the plots.
Page | 125
Figure 3.9
Relative level of C-FABP mRNA in transfectant and LNCaP cell lines
The Real-time PCR products of LNCaP-WT (A), LNCaP-R109A (B),
LNCaP-R109/129A (C), LNCaP-V (D) and LNCaP (LNCaP) were
analyzed by Chromo4 fluorescence detector. The mean and S.D. of
relative C-FABP mRNA level for each cell line from three individual
experiment were shown in the bar chart. LNCaP cells were used as
calibrator.
Page | 126
3.3
Discussion
C-FABP, which was originally isolated from psoriatic skin, is a typical FABP family
member with high affinity to long chain fatty acid. It has been revealed that
overexpression of C-FABP may promote VEGF to facilitate angiogenesis which
contributed to tumor progression such as tumor formation and metastasis [158, 159].
However, the exact mechanism is still not clear. In order to establish transfectants
expressing mutant forms of C-FABP which are not able to bind to fatty acids, point
mutations were introduced to two key amino acids (Arg109 and Arg129) which are
responsible for fatty acid-binding in C-FABP gene. As a result, the relationship between
the fatty acid binding capacity and the tumor promoting function can be assessed by
investigating whether the transfectants expressing mutant C-FABP can still promote
tumor growth.
Two mutated gene have been successfully generated from the wild type C-FABP gene
using Transformertm Site-Directed Mutagenesis Kit: (1) C-FABP-109A where Arginine
109 was converted to Alanine 109; (2) C-FABP-109/129A where Arginine 109 and
Arginine 129 were converted to Alanine 109 and Alanine 129. The point mutations in
both mutated genes were confirmed by sequencing analysis. Then, the wild type and
mutant C-FABPs were transferred into a mammalian expression vector pIRES2-EGFP
with enhanced green fluorescent protein coding region using XhoI and PstI restriction
digestion enzymes. The pIRES2-EGFP empty vector and vectors containing
C-FABP-WT, C-FABP-109A, and C-FABP-109/129A were transfected into LNCaP
cells to create transfected cell lines LNCaP-V, LNCaP-WT, LNCaP-R109A and
LNCaP-R109/129A respectively.
Page | 127
LNCaP cell line, established from a needle aspiration biopsy of the lymph node of male
patient with diagnosed metastatic prostate carcinoma, is a wildly used androgen
sensitive human prostate cancer model cell line [60]. Two properties of LNCaP cells
make them a particularly difficult cell line to generate transfectants compared to other
cell lines --- (1) LNCaP cells have a low growth rate and a tendency to form aggregates;
(2) LNCaP cells have a poor survival rate when seeded at low concentration in tissue
culture plate [166], which can significantly affect the efficiency of transfection as the
cell colonies were developed from a single transfected cell. To overcome the difficulties
in cloning LNCaP cells, the modified transfection method was carried out using a
polyamine based transfection reagent, GeneJammer which provided a higher
transfection efficiency and lower cytotoxity to LNCaP cells compared to calcium
phosphate method [167]. The transfected cell lines were selected by 0.5mg/ml antibiotic
G418 for 10 days in a 75cm2 tissue culture flask. Then, the G418 was removed and
500µl of untransfected LNCaP cells at a concentration of 2x104cells/ml were seeded
into the flask as a feeder layer. The cells were allowed to grow in routine culture
medium until cellular confluence reached at 80%. Then, the cells were re-select
followed the same procedure as for the first selection. After 5 round selections, the cells
were maintained in selective culture medium and successfully transfected colonies were
determined by green fluorescence imaging and recovered by ring cloning.
Page | 128
Transfectant lines: LNCaP-WT transfected with wild type C-FABP, LNCaP-R109A
transfected with single site mutated C-FABP, LNCaP-R109/129A transfected with
double site mutated C-FABP and LNCaP-V (control) transfected with pIRES2-EGFP
vector only were successfully generated. The wild type or mutant C-FABP cDNAs in
each transfected cell line were examined by RT-PCR. The Western Blot assays were
applied to determine the protein level of C-FABP-WT, C-FABP-R109A and
C-FABP-R109/129A in each transfected cell line. The results demonstrated that
expression level of C-FABP-WT in pooled LNCaP-WT cells and mutant C-FABPs in
pooled LNCaP-R109A and LNCaP-109/129A cells were at similar level, which
indicated that these transfectants were suitable for comparison. However, there is no
C-FABP cDNA or protein has been detected in LNCaP cells transfected with empty
vector and paraental cells indicating that transfected with pIRES2-EGFP vector did not
change the expression level of C-FABP. The same pattern of relative C-FABP mRNA
level obtained by Real-time PCR analysis also confirmed the Western Blot results.
The results in this chapter demonstrated the successful establishment of tansfected cell
lines with expression of wild type or mutant C-FABPs. However, the effect of wild type
or mutant C-FABP expression in these transfectants is unknown. These aspects such as
fatty acids uptake ability, cell growth rate, invasion and tumor formation will be
examined and presented in the Chapter 4.
Page | 129
Chapter 4
Effect of overexpression of wild type and
mutant C-FABPs on fatty acids uptake ability
and tumorigenicity in prostate cancer cells
4.1
Introduction
C-FABP is associated with malignant tumor progression as mentioned previously [160].
However its mechanism of action is still not clear. In Chapter 3, four transfected LNCaP
cell lines with expression of wild type and mutant C-FABPs were successfully
established to generate transfectants with different fatty acids uptake capacities. To
reveal whether the fatty acid binding ability is essential for C-FABP to promote cancer
development progression, fatty acids uptake ability of transfected cell lines were
monitored. In this Chapter, experiments were performed to study the effect of reduced
fatty acid binding ability on the tumor-promoting activity of C-FABP.
Long chain fatty acids are not only served as metabolic fuel but also act as endogenous
ligands for nuclear receptors such as PPAR [146]. Within the cells, it is possible that
excessive intracellular fatty acids may contribute to both breast and prostate cancer risk
and disease progression [168-170]. Although the three key amino acids conserved in
FABPs were suggested to be the fatty acid binding site, direct evidence on the effect of
changing this site on fatty acid binding ability of C-FABP is lacking. Thus, it is
important to examine the long chain fatty acids uptake capacities of transfected cell
lines expressing wild type and mutant C-FABPs. The fatty acid uptake assay was
performed to assess relationship between the key amino acids mutation and the fatty
acid binding capability of C-FABP.
Page | 130
Cell proliferation and invasion ability are two prominent features contributory to
tumorigenicity and metastasis. The recent studies showed that cell proliferation rate was
significantly reduced (by 2.5-fold) in siRNA transfected prostate cancer cell line
PC-3M, in which the expression level of C-FABP was suppressed, compared to the
parental cell line [160]. Furthermore, it has been demonstrated that suppression of
C-FABP expression resulted in great reduction of the cell invasiveness and inhibition of
metastasis [159, 160]. On the other hand, Fang et.al reported that overexpression of
C-FABP in transfected human oral squamous carcinoma cells was associated with
promoting the cancer cells growth rate and invasion ability [171]. These findings
suggest that increase expression of C-FABP is related to malignant progression of
these cells.
Tumorigenesis is complex progresses, which involves multiple mutations of tumor
suppress genes or proto-oncogenes resulted in losing control of its cell proliferation.
The studies showed that androgen-dependent human prostate cancer LNCaP cells rarely
formed tumors with 0%-50% tumorigenicity rate when inoculated subcutaneous (SC) in
immunodeficient nude mice [60, 172] and even if the tumor was formed, the tumor
growth was very slow. Morgan et.al showed that suppression of C-FABP expression
level in PC-3M cells significantly reduced tumor formation ability of the cancer cells
both in vitro and in vivo [160]. Similar results had been achieved by using antisense
RNA to suppress C-FABP expression [159]. However, the tumorigenicity of LNCaP
cells with overexpression of C-FABP has not been reported. In contrast to PC-3M cells
which express very high level of C-FABP, the weakly malignant LNCaP cells do not
Page | 131
express detectable level of C-FABP. It is interesting to know whether the tumorigenicity
of LNCaP can be increased by elevated C-FABP expression.
Since the biological activity of C-FABP in normal condition is to bind to fatty acids and
to transport them into cells, it is important to understand whether the ability of binding
to fatty acids is related to the tumorigenicity promoting function of C-FABP in
experiments of Chapter 4. Firstly, the fatty acid uptake capacity of cloned transfectants
LNCaP-WT, LNCaP-R109A, LNCaP-R109/129A and the control transfectant LNCaPV was measured and compared. Then transfectants were subjected to different in vitro
and in vivo assessments to analyze the effect of overexpression of wild type and mutant
C-FABP on the cell properties associated with tumor development and malignancy.
Page | 132
4.2
Results
4.2.1 The effect of increased expression of wild type and mutant of
C-FABPs on cellular fatty acid uptake
The orange-red florescence-labeled fatty acid BODIPY 558/568 C12 {4, 4-difluoro-5(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene-3-dodecanoic acid}, which act as the
analogue of natural LCFA, was applied to examine the level of fatty acid uptake for
each transfectant. The fluorescence intensity of each single cell before and 30mins after
adding labeled LCFA was measured using an EPICS XL Cytometer at the wave length
of 570 nm to assess the transfectants fatty acid uptake capacities as described in
Chapter 2 section 2.2.3.
The fatty acid uptakes in different transfected cell lines were shown in Figure 4.1. The
peaks in the each diagram represented the red fluorescence intensities which were
quantified by manufacture’s software as demonstrated in X-Mean. The number and
proportion of cells in each cell line were also indicated in the diagram column number
and %Gated respectively. Relative levels of LCFA uptake were shown in Figure 4.2.
When the level of fatty acid uptake in LNCaP-V cells was set at 1, levels in LNCaP-WT,
LNCaP-R109A, and LNCaP-R109/129A were 3.1±0.7, 1.7±0.5 and 1.4±0.5
respectively. In comparison with the control transfectant, the fatty acid uptake by
LNCaP-WT cells was significantly higher (Student t-test, P=0.0064). However, the
fatty acid uptake between LNCaP-R109A and the control LNCaP-V, or that between
LNCaP-R109/129A and the control transfectant, was at similar level (Student t-test,
P =0.095, P = 0.216). In addition, no significant difference was observed in fatty acid
uptakes between LNCaP-R109A and LNCaP-R109/129A (Student t-test, P=0.584).
Page | 133
Figure 4.1
Fatty acid uptakes by mutant and wild-type C-FABP cDNA
transfectants at absence and presence of red florescence labeled LCFA.
Flow cytometry analysis of LCFA uptake by different cell lines.
Florescence labeled BODIPY 558/568C12 was added to the cultured cells
and plated at 1x106cells/well in a 6-well plate. The fluorescence intensity
of each transfected cell lines was assessed (X-Mean) and plotted as
histogram before (left panel) and after 30min (right panel) incubation.
A1: LNCaP-WT; A2: LNCaP-R109A; A3: LNCaP-R109/129A and A4:
LNCaP-V.
Page | 134
Figure 4.2
Relative levels of LCFA uptake by mutant and wild-type C-FABP
cDNA transfectants
Relative levels of LCFA uptake by LNCaP-WT (Column 1),
LNCaP-R109A (Column 2) and LNCaP-R109/129A (Column 3) and the
control cells LNCaP-V (Column 4) were measured by flow cytometer.
LNCaP-WT had the highest fatty acid uptake capacity and significantly
higher than the rest transfectants (Student t-test P<0.05). The relative
level of LCFA uptake by LNCaP-R109A was higher than that of
LNCaP-R109/129A and the control tansfectant LNCaP-V but the
differences are not significant (Student t-test P>0.05). Data expressed as
means±SD of three independent experiments.
Page | 135
4.2.2 Effect of wild type and mutant C-FABPs on cell proliferation
To find out whether overexpression of wild type or mutant C-FABPs were correlated
with the cell growth rate, proliferation assays were conducted to assess cell
proliferation. Standard curve of each tested cell line was constructed as described in
section 2.2.4.1. Diagrams in Figure 4.3 presented standard curves for the four
transfectants. The MTT forms crystals in cytoplasm and the crystals were dissolved by
DMSO. The colorimetric measurement was performed at 570nm wavelength using a
spectrophotometer. The values obtained from the plate reader were converted to cell
numbers which were extrapolated from the standard curve.
The detailed proliferation assay results were demonstrated in Figure 4.4. During the first
three days, growth rates of all four transfectant lines were very similar. However, from
the 3rd day on, the proliferation rate of LNCaP-WT started to be higher than those of
other lines. On the 4th and the 5th day, the numbers of LNCaP-WT cells were
44070±1314 and 50508±1193 respectively. Whereas in the same time points, numbers
of cells from the control line were 34445±1044 and 40095±2947 respectively. The
proliferation rate of LNCaP-WT was increased 28% (Day 4) and 26% (Day 5)
compared with LNCaP-V and significantly higher than the control (Student t-test,
P=0.001). On the other hand, cell numbers of LNCaP-R109A and LNCaP-R109/129A
were not significantly different when compared to control transfectant LNCaP-V cells
during the 5 day culture (Student t-test, P=0.482, P=0.055). The final cell counts on day
5 and statistical comparison were summarised in Table 4.1.
Page | 136
Figure 4.3
Standard curves of different transfected cell lines
For each cell line, a standard curve was established by plotting
absorbance (OD at 570nm) (Axis Y) against the number of cells (Axis
X). The cell number of samples was determined by comparing serially
diluted standard from the standard curve. The curve equation and
regression value of standard curve are presented in the diagrams.
Page | 137
Figure 4.4
Time course curve of proliferation rate for wild type and mutant
C-FABPs transfected LNCaP cells
The number of cells for each transfectants, shown as mean of three
individual experiments, were obtained every day during the 5 day period.
Cell lines
Mean number of cells ± SD
P Value
LNCaP-WT
50508 ± 1193
0.001
LNCaP-R109A
41271 ± 1088
0.482
LNCaP-R109/129A
36115 ± 1587
0.055
LNCaP-V
40095 ± 2947
---
Table 4.1
Cell counts of transfectants at the end point of proliferation assay
The final cell counts on day 5 and the statistic analyses were show above.
P values were obtained by comparing sample groups to control LNCaP-V.
Page | 138
4.2.3 Invasiveness of different transfected cell lines
A aodified Boyden Chamber assay was applied to evaluate the invasiveness of different
transfected cell lines. This chemotactic directional migration assay was used wildly to
reveal the cell invasion ability which correlates well with the metastasis potential [173].
The cells were examined in a Boyden Chamber with 6.5mm diameter polycarbonate
membrane coated with 30μg of matrigel to the upper compartment before the cells were
seeded. After 24 hours incubation, cells remaining in the upper compartment of the
filters were removed and the cells attached to the lower side of the filter were fixed and
stained by crystal violet reagent. The numbers of migrated and invaded
cells were
counted under a light microscope at 10/0.25 magnification. On each filter, nine random
fields were counted and the representatives of each transected cell line were shown in
Figure 4.5.
The results were normalized by the cell number after 20 hours routine culture and were
shown in Figure 4.6. All migrated cells remained attaching to the low surface of the
membrane and no cells were found in the culture medium of the low chamber. When the
number of the invaded cells from LNCaP-V was set at 1, the numbers of the invaded
cells from LNCaP-WT, LNCaP-R109A and LNCaP-R109/129A were 2.2±0.3, 1.6±0.2,
and 1.2±0.2 respectively. The number of invaded cells from LNCaP-WT cells was the
highest in all transfected cell lines and more than 2-fold higher than the number of
invaded cells from LNCaP-vector cells (Student t-test, P=0.002). The invasiveness of
LNCaP-R109A cells was higher than LNCaP-R109/129A and LNCaP-vector cells, but
Page | 139
was significantly lower than that of LNCaP-WT cells (Student t-test, P=0.003). The
invasiveness of LNCaP-R109/129A cells was at similar level to that of the control
LNCaP-Vector cells (Student t-test, P=0.247). Difference of invaded cells between
LNCaP-R109A and LNCaP-R109/129A was not significant (Student t-test, P=0.06).
Page | 140
1
2
3
4
Figure 4.5
The images of invaded cells of different transfectants on the lower
filter surface of the Boyden Chamber
The appearances of invaded cells from LNCaP-WT (Panel 1),
LNCaP-R109A (Panel 2), LNCaP-R109/129A (Panel 3) and LNCaP-V
(Panel 4).
Page | 141
Figure 4.6
The invasiveness of cell lines transfected with wild type and mutant
C-FABP genes
The columns represent the invasiveness of LNCaP-WT (Column 1),
LNCaP-R109A (Column 2), LNCaP-R109/129A (Column 3) and
LNCaP-V (Column 4). The invasiveness of LNCaP-WT which expresses
of wild type C-FABP was significantly higher than that of
LNCaP-R109A, LNCaP-R109/129A and LNCaP-V. The invasiveness
was at a downward trand from column 1 to column 4 but no statistical
difference
was
found
between
either
LNCaP-R109A
and
LNCaP-R109/129A or LNCaP-R109/129A and LNCaP-V. The results
are the mean ± SD of three individual experiments.
Page | 142
4.2.4 Soft agar colony formation assay of transfected cell lines
The tumourigenicity of the control and the transfectant cell lines was tested by
examining their anchorage-independent growth in a soft agar assay. The images of soft
agar assay for each transfectant were shown in Figure 4.7 and the results of the soft agar
assays were shown in Figure 4.8. The accurate quantitative assessment and P values of
Student t-test for each transfected cell line compared to control LNCaP-V were
presented in Table 4.2.
The number of colonies produced in the soft agar by LNCaP-V, LNCaP-WT, LNCaPR109A and LNCaP-R109/129A were 130±48, 798±181, 220±66, and 157±38,
respectively. In comparison with the control, the number of colonies produced by
LNCaP-R109A and LNCaP-R109/129A cells was increased slightly but was not
statistically different (Student t-test, P=0.1267, P=0.4855). The most significant change
was observed in LNCaP-WT cells which produced more than 6-fold increase in the
number of colonies formed in soft agar assay comparing to the control. Further analysis
showed that no statistical difference was found between the number of colonies
produced by control LNCaP-R109A cells and the number of colonies produced by
LNCaP-R109/129A cells (Student t-test, P=0.2240). However, the differences between
the colony numbers produced by LNCaP-R109A or LNCaP-109/129A were
significantly reduced when compared to LNCaP-WT (Student t-test, P<0.005).
Page | 143
A
B
C
D
Figure 4.7
The colonies produce by transfectants in soft agar assay
After incubation for four weeks, 3 hours staining by MTT was performed
on colonies developed from LNCaP-WT (Panel A), LNCaP-R109A
(Panel B), LNCaP-R109/129A (Panel C) and LNCaP-V (Panel D) were
displayed above. Cells from each transfected cell lines were tested in
three plates.
Page | 144
Figure 4.8
Colonies formation ability of transfected cell lines in soft agar assay
The number of colonies was counted using GelCount with optimised
setting: Edge detaction: 30; Centre detaction: 50; Colony diameter:
150µm-800µm; Circularity: 60; Density: 0.75.
Cell lines
Mean number of cells ± SD
P Value
LNCaP-WT
798 ± 180.6
0.003
LNCaP-R109A
220 ± 65.7
0.1267
LNCaP-R109/129A
157 ± 38.1
0.4855
LNCaP-V
130 ± 47.5
---
Table 4.2
The numbers of colonies produced by transfectants in soft agar assay
Numbers of colonies (mean ± SD) produced by different transfectants in
three separate soft agar assays were shown above and P values were
obtained by comparing sample groups to control group LNCaP-V.
Page | 145
4.2.5 Expression of wild type C-FABP increased tumourigenicity in vivo
To examine the tumourigenicity of wild type and mutant C-FABPs transfected cell lines,
cells were harvested and injected subcutaneously into the right flank of Balb/C
immuno-incompetent nude mouse as described in Chapter 2 section 2.2.7.
A representative tumor developed inside a mouse from each transfectant group was
displayed in Figure 4.9A and tumor masses resected from the corresponding the tumorbearing animals were also displayed in the lower panel of Figure 4.9A. The appearance
of two representative tumors from mouse inoculated with LNCaP-WT or LNCaP-V
cells were displayed in Figure 4.9B. In addition, the tumor growth curves of all
transfectants were shown in Figure 4.10 according to the average tumor volume
measured and calculated. Finally, the incidence of tumors produced by each transfected
cell lines in nude mice and tumor weight at autopsy were summarized in Table 4.3.
Both Figure 4.9 and Figure 4.10 clearly showed that the size of tumors produced by
LNCaP-WT cells were much bigger than those produced by the rest groups of mice. It
can be noticed in Figure 4.9B that the tumor produced by cells overexpressing C-FABP
appeared dark-reddish, as opposed to a pale appearance of tumors generated by control
transfectant LNCaP-V. The latent period shown in Table 4.3 was different amongst
different mice and ranged from 13 to 25 days, no significant difference was found on
the average length of latent time amongst the different groups. However, the shortest
length of latent period for LNCaP-WT cells was 13 days while for rest transfectants the
shortest length was 18 days. The Table 4.3 also showed that 3 out of 8 (37.5%) mice
Page | 146
inoculated with LNCaP-R109/129A and LNCaP-V cells and 4 out of 8 (50%) mice
inoculated with LNCaP-R109A cells produced tumors. Whereas in the group inoculated
with LNCaP-WT cells, 7 out of 8 (88.9%) mice developed tumors. The volumes of
tumors produced by the four groups of mice at different time points in nude mice were
shown in Figure 4.10. The average volume of tumours produced in the mice inoculated
with LNCaP-WT cells was significant greater than those produced by the mice
inoculated with LNCaP-R109A, LNCaP-R109/129A and LNCaP-V cells respectively.
At autopsy (nine weeks after inoculation), the average weight of the tumours produced
by LNCaP-WT, LNCaP-R109A, LNCaP-R109/129A and LNCaP-V was 614.3±192mg,
217.5±69.5mg, 59.3±19mg and 46.7±15.3mg respectively. While the average weight of
tumours produced by LNCaP-R109A or LNCaP-R109/129A was not significantly
increased (Mann-Witney U-test, P=0.059, P=0.879), the average weight of tumours
produced by the LNCaP-WT cells was significantly heavier than that produced by
LNCaP-V cells by 13.2-times (Mann-Witney U-test, P=0.003).
Page | 147
A
B
Figure 4.9
1
2
The tumor produced by nude mice inoculated with transfected cell lines
(A) Each picture is a representative animal from each group:
LNCaP-WT (Lane 1), LNCaP-R109A (Lane 2), LNCaP-R109/129A
(Lane 3) and LNCaP-V (Lane 4). Tumor masses resected from the
corresponding the animals were also displayed below each Lane
respectively. The tumor produced by LNCaP-WT was pointed out by an
arrow but the tumors produced by the rest transfectants were
imperceptible. (B) The representative tumor produced by LNCaP-WT
(Panel 1) and LNCaP-V (Panel 2).
Page | 148
Figure 4.10
Average tumor volume of each nude mice group inoculated with
different transfected cell lines
Wild type, two mutant and control transfectants were inoculated
subcutaneously
into
right
flank
of
four
groups
of
Balb/C
immuno-incompetent nude mice (8 animals in each group). The tumors
became visualized and measurable after average three weeks latent phase.
Tumor volume was measured every three days and calculated by the
following formula: Length x Width x Height x 0.5236.
Page | 149
Table 4.3
Incidence of tumors produced by transfectants in nude mice and tumor weight at autopsy
a
Tumor incidence is the percentage of the number of mice with tumors/ total number of inoculated animals.
b
Latent period is the number of days from the time of inoculation to the time of first appearance of tumor.
c
Tumor weight was measured at autopsy performed 60 days after inoculation of the transfectant cells.
Page | 150
4.3
Discussion
In the first part of this chapter, the relationship between the tumourigenicity-promoting
function of C-FABP and its fatty acid-binding ability was investigated. As mentioned in
Chapter 2, C-FABP binds to fatty acids through a binding motif which consists of three key
amino acids (Arg109, Arg129, and Tyr131) and replacing any one of these key amino acids
with another will almost completely deprive C-FABP of its fatty acid binding ability [165,
174]. Fatty acid uptake is a strictly regulated process and it is related to the levels of certain
FABPs in LNCaP cells [175]. The fatty acid binding assay revealed that the fatty acid
uptake capacity of LNCaP cells was significantly increased when C-FABP was forced to be
overexpressed. On the other hand, the fatty acid uptake capacities stayed at same level
between either LNCaP cells with expression of single point mutated C-FABP (C-FABPR109A) and control cells (LNCaP) or LNCaP cells with expression of double points
mutated C-FABP (C-FABP-R109/129A) and LNCaP cells. These results indicated that
elevate the expression of C-FABP in LNCaP cells can result in enhancing its fatty acid
uptake capability. Furthermore, it has been also demonstrated that expression of mutated CFABPs generated by site-directed mutagenesis can not increase the fatty acid uptake
capability compared to the control cells, which suggested that the ability of C-FABP to
bind and to transport fatty acids into these transfectant cells depends on the structural
integrity of its fatty acid-binding motif and changing amino acids in this part can result
losing most fatty acid-binding and transporting capability.
Page | 151
In order to investigate whether up-regulation of intracellular fatty acid signaling activities is
associated with tumor malignancy progression, different bioassays were applied to examine
the transfected cells’ biological properties. Bioassays showed that the proliferation rate,
invasiveness and in vitro tumourigenicity of the LNCaP-WT cells, which expressed high
level of wild type C-FABP, capable of up-taking high level of fatty acids, was significantly
higher than those of the LNCaP-V cells. These results indicated that increasing C-FABP
expression was able to promote the malignant progression in LNCaP cells which did not
express C-FABP prior to the transfection. Both LNCaP-R109A and LNCaP-R109/129A
cells, which were generated by C-FABP cDNA with point mutations and have a similar
ability of up-taking fatty acids, exhibited similar levels of invasiveness and tumourigenicity
compared to the control LNCaP-V cells. These results indicated that the tumourigenicitypromoting activity exerted by C-FABP is related to its ability of binding and transporting
fatty acids. When transfected cells’ ability of uptake fatty acids was reduced to the similar
levels to the control cells by double mutations, C-FABP lost its biological function of
promoting tumourigenicity. As a result, LNCaP-R109/129A produced similar number of
colonies as LNCaP-V. Furthermore, when the ability of C-FABP to bind and transport fatty
acids was reduced, but not completely eliminated by a single mutation as observed in
LNCaP-R109A cells, cell growth rate, invasiveness and tumourigenicity is significantly
lower than that of the LNCaP-WT cells, but higher than that of the control cells. It appeared
that the biological function of C-FABP to promote tumor malignant progression was
reduced as its reducing capability of uptake fatty acids which indicated that the increased
uptake of fatty acids played a key role for tumourigenicity promotion.
Page | 152
When inoculated in nude mice, 7 out of 8 (88.9%) mice inoculated with LNCaP-WT cells
developed tumours, but only 3 out of 8 (37.5%) mice inoculated with LNCaP-R109/129A
and LNCaP-V cells respectively produced tumours. At the end of the test, the average
weight of the tumours produced by LNCaP-WT was 5.8- and 7.3- times of those produced
respectively by LNCaP-r109/129a and LNCaP-V cells. This result showed that not only
that the wild type C-FABP-expressing cells produced more but also larger tumours than
those cells either not expressing C-FABP or expressing mutated C-FABP which was not
able to bind and transport fatty acids. In contrast to this finding, our previous work
demonstrated that suppressing expression of C-FABP by either antisense mRNA or RNA
interference in the highly malignant prostate cancer cell line PC-3M has greatly inhibited
its ability of forming tumours in nude mouse [159, 160]. The results of the current and
previous work in nude mouse further confirmed that C-FABP promotes malignant
progression of prostate cancer by its binding and transporting intracellular fatty acids into
the cancer cells.
Page | 153
Chapter 5 Effect of expression wild type and mutant
C-FABPs on VEGF and angiogenesis
5.1
Introduction
Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis which
is essential for growth and expansion of solid tumor [176]. Previous studies demonstrated
that overexpression of C-FABP in rat Rama 37 cell model significantly increased the level
of VEGF mRNA and protein. In addition, the transfected Rama 37 cells with higher
expression of C-FABP and VEGF produced larger tumors and metastases (58.8%) when
compared to the non-metastasis control cells [158]. Recent studies also showed that
suppressed expression of C-FABP in PC-3M cells had greatly reduced their
tumorigenecity, probably through down-regulating the expression of VEGF [159]. These
results suggested a strong positive correlation between C-FABP and VEGF.
Angiogenesis is the process of generating new blood vessel and involves multiple steps:
(1)
Endothelial cells escape from the stable location by breaking through the
basement membrane that envelops the existing blood vessels
(2)
Endothelial cells migrate toward angiogenic stimulates such as wound associated
macrophages or released from cancer cells
(3)
Endothelial cells proliferate to provide certain amount of cells for new vessels
(4)
Endothelial cells reorganize into a tubular structure
Page | 154
Angiogenesis is a vital process in progression of cancer from small, localized neoplasms to
larger, growing tumors. It has been wildly reported that VEGF is a key mediator of
angiogenesis in prostate cancer and the expression level of VEGF in advanced prostate
cancer is significantly increased compared with non-malignant prostate tissue [177, 178].
The significance of angiogenesis in prostate cancer has been well established and
researches showed that angiogenesis has correlated with Gleason score, tumor stage,
metastasis and survival rate in prostate cancer [179-181]. Using the chorioallantoic
membrane (CAM) assay, Jing et.al found that overexpression of C-FABP in Rama37 cells
resulted in an enhanced angiogenic activity in the conditioned medium. On the other hand,
the angiogenesis was inhibited in CAM assay when anti-VEGF antibody was added [158],
indicating C-FABP may promote angiogenesis through up-regulated VEGF expression.
To investigate the possible correlation between the expression level of wild type, mutant
C-FABPs and the expression of VEGF, immunohistochemistry, western blot and
enzyme-linked immunosorbent assays were carried out to analyze the level of VEGF
expression in primary tumors from nude mice, in transfected cell lines and in their
conditioned medium respectively. In this chapter, the angiogenesis abilities of each
transfected cell lines were also assessed.
Page | 155
5.2
Results
5.2.1 Detection of VEGF protein expression in transfectants and their
conditioned medium
Western blot was applied to measure the level of VEGF expression in transfected cell lines
as shown in Figure 5.1A. VEGF expression level in transfected cell lines were quantified
using scanning densitometry as shown in Figure 5.1B. The β-actin expression in each cell
lines was also analyzed as loading control. The results showed that all four transfectant cell
lines expressed VEGF protein (23KDa). Noticeably, VEGF expression in LNCaP-WT was
significantly higher compared to the rest of transfectant cell lines (Students t-test P<0.05).
When the level of VEGF expression in control LNCaP-V cells was set as 1, the level of
VEGF in LNCaP-WT was significantly increased by more than 3-fold (3.48±0.350).
In addition, LNCaP-R109A showed higher VEGF expression (1.71±0.294) compared to
LNCaP-R109/129A (0.97±0.177) but no significant difference has been found when
compared to control cell line LNCaP-V (Students t-test P>0.05).
Page | 156
A
B
Figure 5.1
Expression level of VEGF in LNCaP transfected cell lines
(A) The VEGF expression in LNCaP-WT (Lane 1), LNCaP-R109A (Lane 2),
Rama37 (Lane 3), LNCaP-109/129A (Lane 4) and LNCaP-V (Lane 5) was
analyzed by Western blot. (B) The relative levels of VEGF expression in
different transfectants: LNCaP-WT, LNCaP-R109A, LNCaP-109/129A and
LNCaP-V were shown in column 1, 2, 3 and 4 respectively. The results
(mean±S.D. of three individual experiments) were obtained by densitometry
analysis of the band intensities.
Page | 157
The level of VEGF protein, which was secreted by transfectant cells, in cell culture
supernatant was measured using ELISA analysis and quantified by comparing to a standard
curve, which is obtained from a serial dilution of a known-concentration solution of human
VEGF, as presented in Figure 5.2A. The results in Figure 5.2B demonstrated that the
amount of VEGF protein produced by control transfectant cells LNCaP-V in 100µl
conditioned medium (96.0±23.7pg) was almost the same as that produced by
LNCaP-R109/129A (93.3±5.2pg). The amount of VEGF protein produced in the same
period
of
time
by
LNCaP-R109A
(109.3±31.4pg)
was
slightly
higher
than
LNCaP-R109/129A or LNCaP-V but no statistical difference has been observed either
between LNCaP-R109A and LNCaP-R109/129A or between LNCaP-R109A and
LNCaP-V (Students t-test P>0.05). However, the amount of VEGF protein produced by
LNCaP-WT (452.7±44.1pg) was 4.1, 4.9 and 4.7 times greater than those secreted by
LNCaP-R109A, LNCaP-R109/129A and LNCaP-V cells respectively. The Students t-test
also showed the significant difference between LNCaP-WT and the rest of transfectants
(Students t-test P<0.05). Moreover, the relative difference of VEGF expression in
conditioned medium was greater than the cell lysate but exhibited a similar pattern.
Page | 158
A
B
Figure 5.2
Analysis VEGF protein level in conditioned medium
(A) A standard curve generated for VEGF ELISA analysis. The quantity of
VEGF was determined by comparing serially-diluted concentrations from
the standard curve. (B) The levels of VEGF expression in conditioned
medium of each transfectants - LNCaP-WT, LNCaP-R109A, LNCaP109/129A and LNCaP-V were shown in column 1, 2, 3 and 4 respectively.
Page | 159
5.2.2 Detection of C-FABPs, VEGF and angiogenesis in primary tumor
tissue from nude mice
The expression levels of C-FABPs and VEGF in tumors produced by four groups of nude
mice were examined using immunohistochemistry as shown in Table 5.1 and 5.2
respectively. The microvessels were also stained by anti-CD34 in each tumor section as
shown in Figure 5.3. Representative picture of immunohistochemical stained sections for
each protein was shown in Figture 5.4. The C-FABPs and VEGF expression were
quantified according to the scoring system explained in Charpter 2, Section 2.2.8.4.
Among the cases examined using C-FABP antibody (Table 5.1), all seven (100%) stumor
from LNCaP-WT nude nice group were positively stained. In this group, six out of seven
samples (86%) were moderately or strongly stained and only one tumor (14%) was weakly
stained. Very similar intensities were observed in tumors produced by LNCaP-R109A and
LNCaP-R109/129A cells. All their samples were positively stained by C-FABP antibody.
Most of them were stained moderately or strongly positive (LNCaP-R109A: 75%;
LNCaP-R109/129A: 100%). For tumors removed from LNCaP-V group, on the other hand,
one tumor was unstained and two were weakly stained.
Page | 160
Table 5.1
Immunohistochemical detection of C-FABP in tumors produced by different transfectants
Strained with C-FABP antibody
Tumor Origin
No. of Tumors
-
+
++
+++
Total Positive
No. (%)
No. (%)
No. (%)
No. (%)
No. (%)
LNCaP-WT
7
0 (0)
1 (14)
3 (43)
3 (43)
7 (100)
LNCaP-R109A
4
0 (0)
1 (25)
2 (50)
1 (25)
4 (100)
LNCaP-R109/129A
3
0 (0)
0 (0)
2 (66)
1 (33)
3 (100)
LNCaP-V
3
1 (33)
2 (67)
0 (0)
0 (0)
2 (67)
Immunohistochemical staining in each sample is graded negative (-), weakly positive (+), moderately positive (++) and
strongly positive (+++)
Page | 161
When stained with anti-VEGF antibody (Table 5.2), all seven (100%) tumors from
LNCaP-WT group were positive. In this group, more than half of the samples (57%) were
stained strongly positive, two (29%) were stained moderately positive and only one (14%)
was stained weakly positive. When tumors produced in other groups were stained, most of
the samples were weakly positive. For tumors produced by LNCaP-R109A cells, three out
four were stained (75%) weakly positive and one was moderately positive. For tumors
removed from LNCaP-R109/129A group and LNCaP-V group, all samples (100%) were
stained weakly positive. Strong positive staining was only observed in tumors from
LNCaP-WT group (four out of seven) and no sample had been stained strongly positive in
other groups.
Page | 162
Table 5.2
Immunohistochemical detection of VEGF in tumors produced by different transfectants
Strained with anti-VEGF antibody
Tumors Origin
No. of Tumors
-
+
++
+++
Total Positive
No. (%)
No. (%)
No. (%)
No. (%)
No. (%)
LNCaP-WT
7
0
1 (14)
2 (29)
4 (57)
7 (100)
LNCaP-R109A
4
0
3 (75)
1 (25)
0 (0)
4 (100)
LNCaP-R109/129A
3
0
3 (100)
0 (0)
0 (0)
3 (100)
LNCaP-V
3
0
3 (100)
0 (0)
0 (0)
3 (100)
Immunohistochemical staining in each sample is graded negative (-), weakly positive (+), moderately positive (++) and
strongly positive (+++)
Page | 163
The CD34-related antigen-stained cells were used to assess microvessel density as shown in
Figure 5.3. For each section stained, 8 randomly selected fields were counted. The average
microvessel density per field in tumors produced by LNCaP-WT cells was 4.67±1.15,
compared to 1.67±1.15, 1.33±0.58 and 0.67±0.58 per field in tumors produced by LNCaPR109A, LNCaP-R109/129A and LNCaP-V cells respectively. When the microvessel density
per field in tumors produced by LNCaP-V cells was set as 1, the relative microvessel density in
tumors produced by LNCaP-WT was significantly (Students t-test P<0.05) increased by 7-fold
(7.0±1.73). The relative vessel density in tumors produced by LNCaP-R109A and
LNCaP-R109/129A was also slight increased to 2.5±1.73 and 2.0±0.87 respectively but no
statistical difference has been found when compare to the density in tumors produced by
LNCaP-V (Students t-test P>0.05).
Page | 164
Figure 5.3
The relative microvessel densities of tumors produced by transfectants in
nude mice
The average number of microvessel per field (mean±SD) was calculated from
all tumors in each group. The microvessel density in tumors produced by
LNCaP-V (column 4) was set as 1 and average numbers of microvessel per field
in rest groups were calculated relative to that of tumors produced by LNCaP-V
group. Column 1: LNCaP-WT; Column 2: LNCaP-R109A and Column 3:
LNCaP-109/129A.
Page | 165
1
2
3
A
B
C
D
(Original magnification, ×200)
Figure 5.4
Immunohistochemical detection of C-FABP, anti-VEGF and CD34
expressed in tumors developed in nude mice
Representative samples were immunohistochemically stained with specific
antibodies against: C-FABP (1), VEGF (2) and CD34 (3). Samples in each of
the four rows were removed from tumors produced by LNCaP-WT (A),
LNCaP-R109A (B), LNCaP-109/129A (C) and LNCaP-V (D).
Page | 166
5.2.3 Measurement of angiogenic activity of VEGF produced by wild
type and mutant C-FABPs transfectants
To analysis biological functions of the VEGF produced and secreted by each transfected
cell lines, the endothelial tube formation assay was carried out using the Millipore In
Vitro Angiogenesis Assay Kit which provided a simple model of angiogenesis in which
the induction or inhibition of tube formation by exogenous signals can be easily
monitored. The human umbilical vein endothelial cells (HUVEC) were seeded onto the
surface of the polymerized ECMatrix with the conditioned medium from each
transfectant cells. After 6 hours incubation at 37oC, cells or tubes were visualized by
provided dye as shown in Figure 5.5 and the cell networking structures were quantified
according to the scoring system explained in Charpter 2, Section 2.2.10.3. The average
numerical values of three individual experiments for each transfected cell line were
shown in Figure 5.6.
The HUVEC tube formation ability was strongly promoted by the conditioned medium
from positive control (human VEGF, 10ng/ml) and LNCaP-WT. The HUVEC cells
were able to form well-assembled and complex mesh like structures in the presents of
conditioned medium from positive control and LNCaP-WT (Figure 5.5). For positive
control and LNCaP-WT group, the average numerical value associated with tube
formation pattern was 5 and 4.25±0.83 respectively. In contrast, the HUVEC cells
remained randomly separated without any signs of organization and unable to complete
tube formation when applied with the culture medium only (negative control). For
negative control, the average numerical value associated with tube formation pattern
Page | 167
was 1.25±0.83. The HUVEC cells were begin to migrate and align themselves and some
of them were able to formed capillary tubes in presence of the conditioned medium
from LNCaP-R109A, LNCaP-R109/129A and LNCaP-V but no closed polygons
pattern were observed. For LNCaP-R109A, LNCaP-R109/129A and LNCaP-V group,
the average numerical value associated with tube formation pattern was 2.75±0.44,
2.25±0.83 and 2.5±0.50 respectively. With the treatment of conditioned medium from
LNCaP-WT, HUVEC cells tube formation ability were significantly increased by 1.5-,
1.9-, 1.7- and 3.4-fold compared to the effect of LNCaP-R109A, LNCaP-R109/129A,
LNCaP-V and negative control respectively (Students t-test P<0.05). Differences of
HUVEC cells tube formation ability either between LNCaP-R109A and LNCaP-V or
between LNCaP-R109/129A and LNCaP-V were not significant (Students t-test
P>0.05). It has also been observed that the number of branch points formed in
LNCaP-WT group was higher than that formed in LNCaP-R109A, LNCaP-R109/129A,
LNCaP-V and negative control group.
Page | 168
A
B
C
D
E
F
Figure 5.5
HUVEC cell network formations on ECMatrix with conditioned
medium from different transfected cell lines
Images of HUVEC cell network structure were taken after 6 hours
incubation in endothelia cell growth media with conditioned medium
from different cell lines. Positive control (A), negative control (B),
LNCaP-WT (C), LNCaP-R109A (D), LNCaP-R109/129A (E), and
LNCaP-V (F).
Page | 169
Figure 5.6
Cell network formation values of HUVEC cells with conditioned
medium from different transfected cell lines
The effect of conditioned medium from different transfected cell lines
was assessed and the cell network formation values were quantified
according to the cell network pattern. The values from five random fields
per well were averaged and three individual assays were performed
(mean±SD). Positive control (P), LNCaP-WT (1), LNCaP-R109A (2),
LNCaP-R109/129A (3), LNCaP-V (4) and negative control (N).
Page | 170
In order to determine whether the biological activities of VEGF played an important
role in tube formation, HUVEC cells in conditioned medium from different cell lines
were treated with anti-VEGF in the angiogenesis assay. After 6 hours incubation at
37oC, cells were stained with reagent from Millipore In Vitro Angiogenesis Assay Kit
as shown in Figure 5.8 and the cell network patterns were analyzed as described
previously. With the treatment of anti-VEGF, HUVEC cells in LNCaP-WT conditioned
medium were unable to form polygons pattern and most of them were randomly
separated without any signs of organization. For LNCaP-WT group, the average
numerical value associated with tube formation pattern was 1.75±1.26 which is
significantly reduced by 2.4-fold compared to samples without anti-VEGF (Students
t-test P<0.05). The average numerical values for the rest of the groups including the
negative control stayed as same level as samples without anti-VEGF (Students t-test
P>0.05). No statistical difference was found between the average numerical value of
LNCaP-WT group and that of LNCaP-V group in presence of anti-VEGF (Students
t-test P>0.05).
Page | 171
A
1
2
B
Figure 5.8
Inhibition of cell network formation by anti-VEGF antibody
(A) Images of HUVEC cell network structure were taken after 6 hours
incubation with treatment of anti-VEGF antibody in conditioned medium
from LNCaP-WT (1) and LNCaP-V (2). (B) The cell network formation
values were quantified according to the cell network pattern:
LNCaP-WT (1) LNCaP-R109A (2), LNCaP-R109/129A (3) and
LNCaP-V (4). The values from five random fields per well were
averaged and three individual assays were performed (mean±SD).
Page | 172
5.3
Discussion
The relationship between the tumourigenicity-promoting function of C-FABP and its
ability of binding to and transporting fatty acids were examined and showed that fatty
acid-binding and transporting ability is essential for C-FABP to promote tumor growth
and expansion. However, whether the increased expression of wild type and mutant
C-FABPs are associated with the level of putative target VEGF is still not clear.
In the first part of this chapter, the levels of VEGF expression both in transfected cell
lines and in their conditioned medium were measured by Western blot and ELISA.
These studies demonstrated that VEGF expression in LNCaP cells transfected with
C-FABP was significantly increased compared to VEGF level in cells transfected with
mutant C-FABPs or the vector. These results suggested that increase expression of
C-FABP can elevate the expression level of VEGF in LNCaP cell modle. Our previous
studies results on Rama 37 cell model and prostate cancer cell PC-3M model as
mentioned previously also supported these findings [158, 159]. VEGF, as the most
proangiogenic factor, plays a crucial role on solid tumor development and expansion. It
was reported that the order of VEGF expression in different prostate cancer cell lines as
judged by intensity of immunohistochemical staining was PC-3 (a parental cells of
PC-3M) > Du145 > LNCaP [182]. The malignancy ranking of these prostate cancer cell
lines is in similar sequence as VEGF expression levels. In clinical research, the higher
levels of VEGF expression in plasma were observed in prostate cancer patients
compared with healthy controls. Based on these findings, it has been indicated that
VEGF may facilitate to prostate cancer malignant progression possibly by stimulate
angiogenesis.
Page | 173
In the second part of Chapter 5, the angiogenic function of VEGF produced by different
transfected cell lines was examined. Immunohistochemistry was performed to
investigate the relationship between C-FABP, VEGF and angiogenesis in tumors
produced by nude mice inoculated with different transfected cell lines. The tumors from
LNCaP-WT group, strong expression of VEGF has been detected and microvessel
densities in these tumors were increased 2.8-, 3.5- and 7.0-fold respectively in
comparison to those developed from LNCaP-R109A, LNCaP-R109/129A and
LNCaP-V. These results suggested that higher expression of C-FABP can stimulate
angiogenesis through upregulation of VEGF. The tube formation assay was also carried
out using the Millipore In Vitro Angiogenesis Assay Kit in which the angiogenic
function of VEGF in conditioned medium from each transfected cell lines can be
qualitatively and quantitatively measured. The results showed that tube formation
ability of HUVEC cells with the treatment of conditioned medium from LNCaP-WT
cells were significantly increased by 1.5-, 1.9-, 1.7- and 3.4-fold compared to the effect
of LNCaP-R109A, LNCaP-R109/129A, LNCaP-V and negative control respectively.
On the other hand, the angiogenesis ability of HUVEC cells in LNCaP-WT conditioned
medium was suppressed when co-cultured with anti-VEGF antibody. These results
combined with those reported previously [158, 159] confirmed that the angiogenesis
was indeed produced by VEGF, rather than other possible angiogenesis factors.
Page | 174
Chapter 6
General discussion and future work
Prostate cancer is the most common form of cancer affecting men in the western
countries and the second leading cause of death after lung cancer. In the UK, more than
35,000 men were diagnosed with prostate cancer and around 10,000 men die from
prostate cancer each year. Current treatment of prostate cancer mainly relies on a
combination of androgen ablation, radiotherapy and chemotherapy. However, tumors
often return with more aggressive hormone independent form. So it is important to
improve the understanding of molecular mechanisms involved in prostate cancer
malignant progression, which may lead to new therapies.
The risk factors for prostate cancer include age, ethnicity, family history and diet. It has
been reported that prostate cancer onset has been attributed to a variety of diet factors
ranging from lack of selenium supplementation [183] to vitamin D [184] and high dairy
fat intake was also associated with increased risk of prostate cancer [185-187]. A longterm study demonstrated that high blood levels of trans fatty acids were associated with
an increased prostate cancer risk [188].
This thesis investigated the relationship between the tumourigenicity-promoting
function of C-FABP and its fatty acid-binding ability. Firstly, transfectant cells with
different abilities of binding to fatty acids have been established and the
tumourigenicity of these transfectants were compared to assess whether the reduced
fatty acid-binding capability is associated with the decreased ability of producing
Page | 175
tumours. Like other FABP family proteins, C-FABP binds to fatty acids through a
binding motif which consists of 3 key amino acids (Arg109, Arg129, and Tyr131). Thus we
used site-directed mutagenesis to convert Arg109or Arg109 and Arg129 into Ala109 or
Ala109 and Ala129 to generate two mutant cDNAs and transfected them and wild type
cDNA into the LNCaP cells to generate transfectant cell lines. Realtime PCR
measurement on C-FABP mRNA showed that the C-FABP mRNA level in LNCaP-WT
cells was increased by 137.5-fold comparing with the control cells. Western blot also
confirmed the overexpression of C-FABP in LNCaP-WT cells. From the results
obtained by our previous studies, it was demonstrated that the biological function of
C-FABP is promote malignant progression of cancer cells. In this study, the effect of
high level increment of C-FABP expression on cell malignant properties such as
proliferation rate, invasiveness and anchorage-independent growth as an indication of
tumourigenecicy, was investigated. It is also showed that the mutant C-FABP mRNA
levels expressed respectively in LNCaP-R109A and LNCaP-R109/129A cells were also
increased to the levels similar to that in LNCaP-WT cells. Thus these transfectants are
ideal cell lines used for comparing biological functions exerted by wild type and mutant
C-FABPs.
As mentioned previously, fatty acid uptake is regulated by certain FABPs in LNCaP
cells [175]. Quantitative assessment showed that the relative levels of fatty acid uptakes
in LNCaP-WT, LNCaP-R109A and LNCaP-R109/129A were increased 3.1-, 1.6- and
1.4- fold of that detected in control cells. In another word, in LNCaP-WT,
LNCaP-R109A and LNCaP-R109/129A cell lines, 210%, 60% and 40% more fatty acid
uptakes occurred comparing to that in the control cells. This result showed that
Page | 176
increased wild type C-FABP expression by 137.5 times in mRNA level can result in an
increment in fatty acid uptake to 3.1- time of that in control; whereas one mutation to
the 3 key amino acids in fatty acid binding motif of C-FABP deprived its fatty acid
up-taking ability by 72% (1-60%÷210%). Similarly, two mutations to C-FABP caused
81% reduction in its fatty acid up-taking ability. These results suggested that the
structural integrity of its fatty acid-binding motif is important for the ability of C-FABP
to bind and to transport fatty acids into these transfectant cells and changing one key
amino acid in this part can result losing most fatty acid-binding and transporting ability.
The biological properties of transfected cells with high up-taking level of fatty acids
seem to be much more aggressive when compared to those of control cells, LNCaP-V.
When tested in in vitro assays, the proliferation rate, invasiveness and tumorigenicity of
LNCaP-WT cells with high level of wild type C-FABP expression were significantly
enhanced. However these properties in LNCaP-R109A and LNCaP-R109/129A cells
which express high level of mutated C-FABP, stayed at similar level as those in
LNCaP-V cells. The results obtained from in vivo assay for tumorigenesis have same
the pattern as in vitro assays. When inoculated with LNCaP-WT cells, 88.9% nude mice
developed tumors. On the other hand, only 50%, 37.5% and 37.5% of the mice
inoculated with LNCaP-R109A, LNCaP-R109/129A and LNCaP-V cells respectively
yielded tumuors. The average tumor weight produced in LNCaP-WT group was also
increased more than 13-times to 614.3±192mg compared that in LNCaP-V group
(46.7±15.3mg). These data showed that increase the ability of up-taking fatty acids in
LNCaP cells by elevate C-FABP expression was able to promote the proliferation,
invasiveness and tumorigenicity both in vitro and in vivo. It has also been demonstrated
Page | 177
that the function of C-FABP to stimulate tumourigenicity was reduced when its
up-taking fatty acids capability was decreased. Taken together, these findings indicated
that the increased up-taking of fatty acids played an important role in tumourigenicitypromotion. Our previous work demonstrated that suppressing expression of C-FABP in
the highly malignant prostate cancer cell line PC-3M has inhibited its ability of forming
tumours in nude mouse [189, 190]. The results of the current and previous work further
confirmed that C-FABP promotes malignant progression of prostate cancer depending
on its binding and transporting intracellular fatty acids capacity.
Angiogenesis plays an important role in growth, malignant progression and metastasis
by promoting endothelial cell proliferation, invasion and capillary differentiation [191193]. In addition, Weidner et.al has reported that the pathological surrogate for
angiogenesis (microvessel density) is correlated with malignancy of prostate cancer
[130]. The current studies showed that angiogenesis-associated genes such as VEGF
significantly increased the prostate cancer risk. The results in chapter 5 showed that
increased expression of biologically active VEGF was detected in wild type C-FABP
transfectants, but not in the mutant types of the transfectant cells.
These results
suggested that increased VEGF expression may be caused by wild type C-FABP and
that the ability of C-FABP to increase VEGF depends on its fatty acid-binding ability.
Fatty acids appeared to play important roles. Fatty acids were identified as signalling
molecules [194] which may be recognised by their nuclear peroxisome proliferatoractivated receptors (PPARs), particularly PPARβ/δ [32].
Page | 178
According to the results in this study and those reported by some other groups, it is
reasonable to hypothesis that there may be fatty acid-initiated signalling pathways
leading to malignant progression in prostate cancer cells. The route of these pathways
may be as following: the elevated expression of C-FABP may give rise to an increased
total uptake of fatty acids and enhanced the possible fatty acid signalling activity. Thus,
it may be possible that excessive levels of free fatty acids are transported by C-FABP
into the nucleus of the cancer cells to activate some as yet unknown mechanisms which
might initiate a chain of molecular reactions resulted in promoting cancer growth and
expansion. One of such actions regulated through this unknown mechanism is to
up-regulate some important down-stream “cancer-promoting” genes, such as VEGF
[164, 189], and hence contribute to carcinogenesis. Thus C-FABP which acted as an
intracellular fatty acids transporter may play a key role during this process. A schematic
hypothesis for the involvement of C-FABP in cancer malignant progression is shown in
Figure 8.1.
Page | 179
Figure 8.1
Model for possible C-FABP signaling pathways in cancer malignant
progression
Page | 180
As a first step of testing this hypothesis, we investigated the relationship between the
tumourigenicity-promoting function of C-FABP and its ability of binding to and
transporting fatty acids in this study and demonstrated that fatty acid-binding and
transporting ability is essential for C-FABP to promote tumor growth and expansion. To
fully establish this proposed route of fatty acids signaling pathways, the future work
should be performed to study the detailed molecular mechanisms on how VEGF is
up-regulated? Effort is also needed to study whether there are any other cancerpromoting molecules up-regulated by this mechanism?
Page | 181
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