View
2
Download
0
Category
Preview:
Citation preview
CCHHAAPPTTEERR 11
RREEVVIIEEWW OOFF LLIITTEERRAATTUURREE
TABLE OF CONTENTS
1.1. INTRODUCTION
1.2. IMMUNE SYSTEM AND CANCER
1.2.1. Innate Immunity
1.2.1.1. Natural killer cells
1.2.1.2. Macrophages
1.2.1.3. Complement system
1.2.2. Adaptive Immunity
1.2.2.1. T lymphocytes
1.2.2.2. B lymphocytes
1.2.3. Tumour escape mechanisms
1.2.3.1. Recognition and selection
1.2.3.2. Downregulation of the immune response
1.2.3.3. Tolerance induction
1.2.3.4. Immunodeficiency in cancer patients
1.3. INFLAMMATION AND CANCER
1.3.1. Major mediators linking inflammation and cancer
1.3.1.1. Cytokines
1.3.1.1.1. Tumour Necrosis Factor- α (TNF-α)
1.3.1.1.2. Interleukin- 1β (IL-1β)
1.3.1.1.3. Interleukin-6 (IL-6)
1.3.1.1.4. Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF)
1.3.1.1.5. Interferons (IFNs)
1.3.1.1.6. Interleukin-2 (IL-2)
1.3.1.1.7. Chemokines
1.3.1.2. Cyclooxygenase-2 (COX-2) and prostaglandins
1.3.1.3. Nitric Oxide (NO) and Nitric Oxide Synthase (iNOS)
1.3.1.4. STAT3
1.4. TUMOUR METASTASIS
1.4.1. Metastatic cascade
1.4.1.1. Detachment of cells from primary tumour
1.4.1.1.1. Adhesion molecules and receptors
1.4.1.2. Tumour cell invasion
1.4.1.2.1. Attachment to ECM
1.4.1.2.2. Degradation of ECM
1.4.1.2.2.1. Protease: Serine protease, cathepsins & MMPs
1.4.1.2.2.1.1. Matrix Metalloproteinases (MMPs)
1.4.1.2.2.1.1.1. Regulation of MMPs
1.4.1.2.2.1.1.2. Tissue inhibitors of metalloproteinases (TIMPs)
1.4.1.2.3. Migration
1.4.1.3. Intravasation
1.4.1.4. Cancer cells in the blood stream
1.4.1.5. Extravasation
1.4.1.6. Implantation into target organ: Specificity of metastasis
1.4.1.7. Tumour angiogenesis
1.4.2. Metastasis suppressor genes
1.5. TUMOUR ANGIOGENESIS
1.5.1. Angiogenic switch
1.5.2. Angiogenic factors
1.5.2.1. Vascular endothelial growth factor
1.5.2.1.1. Vascular endothelial growth factor receptor (VEGFR)
1.5.2.2. Basic fibroblast growth factor (bFGF)
1.5.2.3. Angiopoietins
1.5.2.4. Platelet-derived endothelial cell growth factor (PD-ECGF)
1.5.2.5. Chemokines
1.5.2.6. Transforming growth factor-β
1.5.2.7. Tumour Hypoxia
1.5.3. Naturally occurring angiogenic inhibitors
1.5.3.1. Thrombospondin-1
1.5.3.2. Interferon
1.5.3.4. Metalloproteinase Inhibitors
1.5.4. Tumour-derived inhibitors
1.5.4.1. Angiostatin
1.5.4.2. Endostatin
1.5.5. Pharmacologic agents that inhibit angiogenesis
1.5.5.1. TNP-470
1.5.5.2. Thalidomide
1.5.5.3. CAI
1.5.5.4. Troponin
1.6. APOPTOSIS
1.6.1. Morphology of Apoptosis
1.6.2. Apoptotic Pathways
1.6.2.1. The Extrinsic Pathway
1.6.2.2. The Intrinsic Pathway
1.6.2.3. Perforin/granzyme Pathway
1.6.2.4. Execution Pathway
1.6.3. Bcl-2 family
1.6.4. Caspases – the executioners of apoptosis
1.6.5. Role of p53
1.7. NF-κB
1.7.1. NF-κB-activation pathways
1.7.2. NF-κB and cancer
1.8. CURRENT THERAPIES
1.9. IMMUNOMODULATION BY NATURAL PRODUCTS
1.10. CHEMOPREVENTION BY NATURAL PRODUCTS
1.10.1. Vernonia cinerea Less
1.11. CHEMOPREVENTION BY NATURALLY OCCURRING TERPENOIDS
1.11.1. Vernolide-A
1.11.2. Nomilin
1.11.3. Oleanolic acid
1.11.4. Perillic acid
1
1.1. INTRODUCTION
Cancer is a disease characterized by uncontrollable, irreversible, independent,
autonomous, uncoordinated and relatively unlimited and abnormal over growth of
tissues. It is one of the leading causes of death in the World. According to the
International Agency for Research on Cancer (IARC), in 2002, cancer killed > 6.7
million people around the world; another 10.9 million new cases were diagnosed;
and at the current rate, an estimated 15 million people will be diagnosed annually by
2020. Tumours can be of 2 major types: Benign tumours and malignant tumours.
Benign tumours are generally slow growing expansive masses often with a “Pushing
margin” and enclosed within a fibrous capsule. Malignant tumours are usually
rapidly growing, invading local tissue and metastases to distant sites.
1.2. IMMUNE SYSTEM AND CANCER
The immune system is a remarkably versatile defense system that has been evolved
to protect animals from invading pathogenic microorganisms and cancer. It can
generate an enormous variety of cells and molecules capable of specifically
recognizing and eliminating an apparently limitless variety of foreign invaders
(Kuby, 1997). First, it can protect the host from virus-induced tumours by
eliminating or suppressing viral infections. Second, the timely elimination of
pathogens and prompt resolution of inflammation can prevent the establishment of
an inflammatory environment conducive to tumourigenesis. Third, the immune
system can specifically identify and eliminate tumour cells on the basis of mutations
and expression of stress ligands. This final process is referred to as tumour immune
surveillance, whereby the immune system identifies cancerous or pre-cancerous cells
and eliminates them before they can cause harm. However, tumours can, and do,
develop in the presence of a functioning immune system, and the theory of tumour
immune surveillance has recently been updated by emergence of the newer concept
of tumour ‘immunoediting’ (Smyth, 2007). Immune system can recognize tumour
cells as dangerous and attack them by different means involving different effector
cells. Key-players are NK cells of the innate and T cells of the adaptive arm of the
immune system.
There are two types of immunity: innate and adaptive immunity. Innate
immunity includes all the natural defenses of the human body, it is nonspecific, the
2
first line of defense, and seen clinically as an inflammatory response (Solomon and
Komanduri, 2001).
1.2.1. Innate Immunity
The major components of this system include complement proteins, phagocytes, and
NK cells. The complement proteins consist of plasma proteins and a proteolytic
enzyme cascade, which attacks the cell membrane of the invading microbe and
causes cell lysis. The differences between the carbohydrates produced by
mammalian cells and by bacteria are detected by phagocytes and trigger a pseudopod
response. This system has three major types of phagocytes: (1) neutrophils; (2) cells
of the mononuclear phagocyte system, monocytes in the blood and macrophages in
connective tissue; and (3) organ-specific phagocytes in the liver, spleen, lymph
nodes, lungs and brain (Fox, 2002).
1.2.1.1. Natural killer cells
Natural killer (NK) cells are part of the innate immune system and important players
in the first line of defense against diseases, including malignancies. In contrast to
cells of the adaptive immune system (e.g., T-cells), immunization is not required to
trigger NK cell cytotoxicity. NK cells therefore provide an immediate natural
response. Their rapid cytolytic action and broad target range suggest that NK cells
may be promising candidates for cancer cell therapy with the potential to target a
wide range of malignancies. Research during the past decade has focused on the
identification of the cell surface receptors and effector molecules that NK cells use in
target-cell recognition and destruction. Attention now turns to determine both the
role of NK cells in vivo in innate immunity and their contribution to adaptive
immunity (Hamerman et al., 2005).
1.2.1.2. Macrophages
Macrophages are major immune cells in the innate immune system. They can
function as antigen-presenting cells and interact with T lymphocytes to modulate the
adaptive immune response. The activation of macrophages plays a key role in
inflammatory responses to infection with pathogens. Macrophages can kill pathogens
directly by phagocytosis and indirectly via the secretion of various pro-inflammatory
cytokines such as TNF- α, IL-1β, and IL-6 (Nathan, 1992; Moncada, 1999). When
activated they inhibit the growth of a wide variety of tumour cells and
3
microorganisms. Macrophages may recognize some tumour antigens leading to
tumouricidal activity. The mechanisms of tumour cell killing by macrophages is the
same as with infectious microorganisms through the intra- or extracellular release of
lysosomal enzymes, reactive oxygen intermediates, and nitric oxide.
1.2.1.3. Complement system
Complement is a system of plasma proteins that can be activated by antibody leading
to a cascade of reactions that occurs on the surface of pathogens and generates active
components with various effector functions. Complement proteins are responsible
for cell lysis and mediation of inflammation and enhanced phagocytosis. It is a chief
component of both innate and humoral branch of immune system and is of
substantial relevance in tumour cell destruction (Carroll, 2004). Complement system
is activated by three different pathways: classical, alternative and lectin, leading to a
cascade of reactions that ultimately results in the induction of inflammatory
responses, phagocyte chemotaxis, opsonization, as well as target cell lysis (Kuby et
al., 2002). Intrinsic interactions among the complement activation products and cell
surface receptors provide a basis for the regulation of both B and T cell responses.
1.2.2. Adaptive Immunity
Adaptive immunity functions mainly with the help of lymphocytes. Although innate
and adaptive immunity work through different mechanisms, they are closely linked,
with the adaptive system amplifying the response initiated by the innate system.
Adaptive immunity has some key characteristics: diversity, specificity, and memory.
The system is diverse because it is able to recognize a vast number of foreign
antigens; it is specific because the lymphocytes store a large number of antigenic
specifics; and it has memory because once it has mounted an antigen-specific
reaction, the proliferation and differentiation of the antigen-specific antibodies can
respond much quicker to a second attack by the same antigen (Solomon and
Komanduri, 2001). The two major classes of lymphocytes mediating the adaptive
immune response are T and B lymphocytes.
1.2.2.1. T lymphocytes
T lymphocytes are mononuclear, nongranular leucocyte that matures in thymus and
brings about cell mediated immunity. They are thymus dependent cells and mature
under the influence of thymic hormones. Antigen-presenting cells identify a foreign
4
antigen, process and present it to T lymphocytes. This process is assisted by the
secretion of chemokines, which are chemical attractants that increase the possibility
of the antigen encountering the appropriate lymphocyte (Fox, 2002). After being
programmed in this way, T lymphocytes seek out target antigens. T lymphocytes
have three subtypes. Killer T cells are identified by a surface molecule, CD-8. They
destroy body cells that harbour foreign molecules either from invading organisms or
cells that have undergone a malignant transformation, as well as molecules never
before presented to the immune system. Helper T lymphocytes (with a CD-4 surface
molecule) and suppressor T lymphocytes (with a CD-8 surface molecule) regulate
the immune response of B lymphocytes and killer T cells. The helper T lymphocytes
increase the response of the B and killer T lymphocytes, whereas the suppressor T
lymphocytes decrease this response (Fox, 2002).
1.2.2.2. B lymphocytes
B lymphocytes mature in bursa of Fabricius or bone marrow and brings about
humoral immunity. They are present in blood and lymph and secrete specific
antibodies (or immunoglobulins) when exposed to an antigen, therefore providing
humoral immunity. When the resting B lymphocyte with antibodies on its surface is
exposed to a specific antigen, a reaction is triggered and the B lymphocytes are
activated to differentiate into memory and plasma cells. Plasma cells actively secrete
antibodies into the circulation. A single B lymphocyte produces many daughter cells
and all produce the same antibody. Therefore, the immunoglobulin secreted by a
particular group of B cells is named monoclonal antibody. The memory cells are
very similar to the original B cell and are important in active immunity. They divide
rapidly and produce a clone. When the body is exposed to the same antigen again,
these memory cells are able to mount a secondary response much faster, preventing
disease (Fox, 2002; Solomon and Komanduri, 2001; Mautner and Huang, 2003).
1.2.3. Tumour escape mechanisms
According to the immune surveillance hypothesis, the expression of tumour antigens
during neoplastic transformation would induce an immune response that can control
tumour growth (Burnet, 1970). Several mechanisms have now been recognised that
allow tumours to evade an intact and otherwise competent immune system.
5
1.2.3.1. Recognition and selection: Naturally occurring tumours are not
monoclonal. An effective tumour recognition and cytotoxicity therefore constitutes a
selective pressure towards cells that escape from the immune response. Thus,
tumours have been found to develop in such a way that previously recognised
antigens are no longer expressed, so-called antigen- loss variants (Uyttenhove et al.,
1983; Jager et al., 1996).
1.2.3.2. Downregulation of the immune response: Half of the cancers were
recently found to defend themselves against apoptosis by the local release of
inhibitory molecules, (e.g. TGFb) (Torre-Amione et al., 1990) and by expression of
Fas Ligand (FasL) on the cell surface (Hahne et al., 1996).
1.2.3.3. Tolerance induction: Absence of costimulatory molecules on tumours can
even prevent cytotoxic activation of CTL previously present (Speiser et al., 1997;
Wick et al., 1997), and may involve multiple factors (Habicht et al., 1995). Finally,
tumours may fail to provide the optimal ‘danger’ signaling microenvironment and
associated cytokines, which optimise the function of immune effector cells.
1.2.3.4. Immunodeficiency in cancer patients: This might have to do with
malnutrition, immunosuppressive therapies, but also with other, not yet identified,
factors.
1.3. INFLAMMATION AND CANCER
Chronic inflammation is involved in the pathogenesis of most common cancers. The
etiology of the inflammation is varied and includes microbial, chemical and physical
agents. Many of the factors involved in chronic inflammation play a dual role in the
process, promoting neoplastic progression thereby facilitating cancer prevention
(Mueller and Fusenig, 2004). Approximately, 25% of all cancers are somehow
associated with chronic infection and inflammation (Hussain and Harris, 2007).
Although inflammation acts as an adaptive host defense against infection or injury
and is primarily a self-limiting process, inadequate resolution of inflammatory
responses often leads to various chronic ailments including cancer (Jackson and
Evers, 2006; Schottenfeld and Beebe-Dimmer, 2006). A comprehensive
understanding of the molecular and cellular inflammatory mechanisms involved is
vital for developing preventive and therapeutic strategies against cancer (Hold and
El-Omar, 2008).
6
1.3.1. Major mediators linking inflammation and cancer
Among the major molecular players involved in the inflammation-to-cancer axis, the
notable members are cytokines, COX-2, prostaglandins, iNOS, NO, and STAT3
(Kundu and Surh, 2008).
1.3.1.1. Cytokines
Cytokines, originally known as immuno-regulatory proteins. They are low molecular
weight extracellular signaling proteins secreted by immune and inflammatory cell
populations, which affect growth, differentiation, and viability of cells. Generally,
cytokines are synthesized and secreted rapidly in response to an antigenic stimulus.
Cytokines may be divided into six groups: interleukins, colony-stimulating factors,
interferons, tumour necrosis factor, growth factors, and chemokines (Desai, 2007).
1.3.1.1.1. Tumour Necrosis Factor- α (TNF-α)
TNF-α is a 157 amino acid cytokine and is produced in response to injury and
inflammatory or infectious stimuli by macrophages, lymphocytes, neutrophils, and
structural cells, including fibroblast, smooth muscle cells, astrocytes and microglia.
It is considered as a proinflammatory molecule, augmenting the immune response to
help speed-up the elimination of pathogens and the resolution of the inflammatory
challenge (Brietzke and Kapczinski, 2008). TNF-α has several effects, including
cytotoxicity, antiviral activity, transcription factor activation, and immune response
regulation. TNF-α exerts its effects by binding to specific receptors named TNF-R1
and TNF-R2. TNF-R1 mediates many actions of TNF-α, including cytokine
production, activation of transcription factors like NF-κB, and apoptosis (Bhardwaj
and Aggarwal, 2003).
1.3.1.1.2. Interleukin- 1β (IL-1β)
Several inflammatory interleukins have been linked with tumourigenesis, which
suggests that inflammation is associated with cancer development. These
interleukins include IL-1, IL-6, IL-8, and IL-18. Interleukins mediate different steps
in the pathway leading to tumourigenesis. IL-1β is one of the pro-inflammatory
cytokines belonging to the IL-1 family. Together with other pro-inflammatory
cytokines such as tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and
interleukin- 8 (IL-8), IL-1β can induce acute inflammation (cytokine cascade, high
fever, C-reactive protein (CRP) production) in response to infection. A low
7
concentration of IL-1β has been shown to induce local inflammatory responses
followed by activation of protective immune response, while a high concentration of
IL-1β leads to inflammation-associated tissue damage and tumour invasiveness
(Apte and Voronov, 2002). Autocrine production of interleukin IL-1β promotes
growth and confers chemoresistance in pancreatic carcinoma cell lines (Arlt et al.,
2002). IL-1β secretion into the tumour milieu also induces several angiogenic factors
from tumour and stromal cells that promotes tumour growth through hyper
neovascularization in lung carcinoma growth in vivo (Saijo et al., 2002).
1.3.1.1.3. Interleukin-6 (IL-6)
IL-6 is another major proinflammatory cytokine that participates in inflammation-
associated carcinogenesis (Rose-John and Schooltink, 2007). IL-6 acts as a paracrine
growth factor for multiple myeloma, non-Hodgkin’s lymphoma, bladder cancer,
colorectal cancer, and renal cell carcinoma (RCC) (Aggarwal et al., 2006). IL-6
modulates the expression of genes involved in cell cycle progression and inhibition
of apoptosis, primarily via the JAK-STAT signaling pathway (Lin and Karin, 2007).
An elevated level of IL-6 has been implicated in the pathogenesis of various cancers
(Cozen et al., 2004; Kai et al., 2005; Schneider et al., 2000). The serum levels of IL-
6 have found to be significantly increased and positively correlated with tumour
burden in colon cancer patients (Chung and Chang, 2003). Likewise, IL-6 stimulated
the anchorage-independent growth of human colon carcinoma cells, suggesting its
potential role in tumourigenesis (Schneider et al., 2000). IL-6 is essential for ras-
driven tumourigenesis (Quintanilla et al., 1986). IL-6 contributes to the growth of
cholangiocarcinomas by decreasing promoter methylation of EGFR and by
upregulating growth promoting genes (Wehbe et al., 2006).
1.3.1.1.4. Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF) Granulocyte monocyte-colony stimulating factor (GM-CSF) is a pleiotropic cytokine
secreted by activated CD4+, CD8+ T cells, NK cells NKT cells and dendritic cells
and known to elicit powerful immune responses when combined with gamma
irradiated tumour cell vaccines, in various murine models. GM-CSF modulates the
function of differentiated white blood cells. For example, GM-CSF stimulates
macrophages for antimicrobial and antitumour effects. The cytokine further enhances
healing and repair by its actions on endothelial cells, fibroblasts and epidermal cells.
GM-CSF is the pivotal mediator of the maturation and function of dendritic cells, the
8
most important cell type inducing primary T-cell immune responses. GM-CSF
activates dendritic cells, macrophages, granulocytes and NKT cells thereby
improving tumour antigen presentation (Gillessen et al., 2003). However, excessive
amounts of this growth factor could exert immunesuppressive effects (Bronte et al.,
1999).
1.3.1.1.5. Interferons (IFNs)
Interferons (IFNs) are a large family of multifactorial secreted protein with wide
array of functions including antiviral, antiproliferative, antiangiogenic, apoptotic etc.
IFNs are divided into three groups α, β and γ on the basis of their distinct properties.
They are produced by T cells, NK cells, NKT cells and to a lesser extent by dentritic
cells, macrophages and have shown to mediate pleiotropic effects in both innate and
adaptive immunity (Dranoff, 2004, Goodbourn et al., 2000). IFN-γ signaling
pathways lead to apoptosis and the expression of immune function protein that could
co-operate with T cells in the destruction of tumour (Blanck, 2002). IFN-γ play a
central role in governing immune cell response and T cell proliferation that can
modulate chemokine production and leukocyte recruitment in response to
proinflammatory cytokine (Robson et al., 2001).
1.3.1.1.6. Interleukin-2 (IL-2)
Interleukin-2 was first identified in 1975 as a growth-promoting factor for bone
marrow-derived T-lymphocytes (Morgan et al., 1976). Since then, the spectrum of its
known biological activities has expanded considerably. IL-2 regulates growth and
differentiation of T and B lymphocytes, promotes growth of NK cells and enhances
their cytolytic functions, and is also known to function in some non-lymphoid cells
(Jiang et al., 2003; Gattinoni et al., 2006). It is essential for the maintenance of
peripheral self tolerance as well (Furtado et al., 2002). IL-2 is of clinical value for
stimulating the natural immunity by stimulating natural killer cell and cytotoxic T
lymphocyte production (Neville et al., 2001; Yamaguchi and Sakaguchi, 2006). Mice
deficient in either IL-2 or component of the IL-2 receptor spontaneously develop
lymphoproliferative and autoimmune disease (Furtado et al., 2002).
1.3.1.1.7. Chemokines
Chemokines are a family of proteins that have pleiotropic biological effects.
Chemokines can play several roles in cancer progression, including angiogenesis,
9
inflammation, cell recruitment, and migration, and have a well-known role in
regulating the recruitment and trafficking of leukocytes to sites of inflammation
(Aggarwal et al., 2006). Chemokines are classified as four major groups, i.e., CXC,
CC, XC and CX3C primarily based on the positions of conserved cysteine residues
(Balkwill and Mantovani, 2001; Lu et al., 2006; Allen et al., 2007). In chronic
inflammation, chemokines are usually produced by proinflammatory cytokines. The
central role of chemokines is to recruit leukocytes to the site of inflammation (Lu et
al., 2006). Most tumour cells can produce CXC and CC chemokines, which again
differ in selectivity for particular leukocytes. While lymphocytes represent a
common target of both CXC and CC, neutrophils are targeted only by CXC
chemokines. CC chemokines can also act on other leukocyte subtypes, such as
monocytes, eosinophils, dendritic cells and natural killer cells (Balkwill and
Mantovani, 2001). Like other cytokines, chemokines also act by interacting with
specific receptors expressed by both infiltrated leukocytes and tumour cells in an
autocrine or a paracrine fashion (Balkwill and Mantovani, 2001; Lu et al., 2006).
Several studies have reported the involvement of chemokines and chemokine
receptors in cell proliferation, migration, invasion and metastasis of different types
of tumours (Kundu and Surh, 2008).
1.3.1.2. Cyclooxygenase-2 (COX-2) and prostaglandins
COX-2 promotes the breakdown of arachidonic acid to produce a series of
prostaglandins, which are key mediators of inflammatory responses (Surh et al.,
2001). PGE2 promotes cell proliferation and tumour-associated neovascularization,
and inhibits cell death, thereby favouring tumour growth (Mann and DuBois, 2004).
Several studies have demonstrated that PGE2 is capable of promoting mouse skin
and colon carcinogenesis (Furstenberger et al., 1989; Narisawa et al., 1987). In
response to various external stimuli, such as proinflammatory cytokines, bacterial
LPS, UV, ROS and phorbol ester, COX-2 is transiently elevated in certain tissues
(Surh and Kundu, 2007). Abnormally elevated COX-2 causes promotion of cellular
proliferation, suppression of apoptosis, enhancement of angiogenesis and
invasiveness, etc., which account for its oncogenic function (Surh et al., 2001).
Aberrant induction of COX-2 has been implicated in the pathogenesis of various
types of malignancies (Kundu and Surh, 2008).
10
1.3.1.3. Nitric Oxide (NO) and Nitric Oxide Synthase (iNOS)
Another important inflammatory mediator linking chronic inflammation and cancer
is NO, which is produced endogenously during arginine metabolism by different
isoforms of NOS (Moncada et al., 1991). During inflammation, induced expression
of iNOS in macrophages and epithelial cells leads to production of NO. The
expression of iNOS and the level of NO have been shown to be elevated in various
precancerous lesions and carcinomas (Chen and Stoner, 2004; Jaiswal et al., 2001).
Study demonstrated that topical application of phorbol ester induced iNOS
expression and subsequent NO production, which in turn induced COX-2 expression
via NF-κB activation in mouse skin (Chun et al., 2004). In response to inflammatory
cytokines (e.g., TNF-α and IL-1β) or other inflammatory stimuli (e.g., phorbol ester,
UVB, LPS and DSS), iNOS is transactivated by some transcription factors including
NF-κB (Surh et al., 2001; Yoshida et al., 2000).
1.3.1.4. STAT3
STAT3 (signal transducers and activators of transcription 3) signaling is also a major
oncogenic pathway in epithelial cells and is thought to underpin the pro-malignant
activity of IL-6 (Aggarwal et al., 2006). However, the role of STAT3 in the
regulation of immune cells, and its involvement in tumour-induced immune-
suppression are important for carcinogenesis (Yu et al., 2007). It is well established
that signaling through STAT3 inhibits pro-inflammatory cytokine production and
leukocyte recruitment, but this could be attributed to either IL-6 or IL-10 signaling.
Blockade of STAT3 in tumours increased pro-inflammatory mediator production and
leukocyte recruitment, but was associated with increased cytotoxicity and tumour
regression, suggesting that STAT3 activation blocks antitumour immune responses
(Kortylewski et al., 2005).
1.4. TUMOUR METASTASIS
Cancer is a hyperproliferative disorder recognised to be a second major health
problem in the world. Tumour metastasis is the leading cause of cancer- related
morbidity and mortality. Invasion and metastasis are hallmarks of tumour
malignancy and major obstacles to curative cancer therapies. Metastasis is the
process by which a tumour cell leaves the primary tumour, travels to a distant site via
the circulatory system, and establishes a secondary tumour. Cancer metastasis
11
consists of multiple, complex, interacting, and interdependent steps, requiring
invasion from the primary tumour, intravasation, survival, arrest and extravasation of
the circulatory system and colonization at a distant site. Each step in the process is
rate-limiting; failure to complete any one prevents the tumour cell from producing a
metastasis (Palmieri et al., 2007, Ellis and Fidler, 1996), but the majority of the
tumour cells fail to survive and establish a metastatic cascade, which is termed
metastatic inefficiency.
1.4.1. Metastatic cascade
To successfully colonize a distant organ, a tumour cell must complete all steps of the
cascade. Failure to complete any step results in the failure to colonize metastatic
target organs. Major steps involved in the cascade are as follows
1.4.1.1. Detachment of cells from primary tumour
Detachment of cells of the primary tumour and their adhesion on a distant site are
considered to be the crucial steps in the metastatic cascade. Tumour cells often show
a decrease in cell-cell and/or cell-matrix adhesion. An increasing body of evidence
indicates that this reduction in cell adhesion correlates with tumour invasion and
metastasis (Cavallaro and Christofori, 2001). Cancer cells carry rich content of N-
acetyl neuraminic acid (Neu5Ac or NANA) on the surface, exposing high negative
charge that reduces cell-cell adhesion. Metastatic cancer cells often express a high
density of sialic acid-rich glycoproteins. Cancer cells secrete some powerful
proteolytic enzymes (protease) that reduce cell-cell adhesion which leads to the cell
detachment from primary tumour, evidence that protease inhibitors can block the
cancer cell detachments (Meyer et al., 1983).
1.4.1.1.1. Adhesion molecules and receptors
Cell adhesion is a vital process, essential for the establishment and maintenance of
tissue architecture and differentiation. Cell adhesion molecules (CAMs) are cell
surface glycoproteins that mediate the physical interactions between adjacent cells
and between cells and the surrounding extracellular matrix. CAMs belong to
different protein families, depending on their structural and functional properties
(Francavilla et al., 2009). They are involved in the regulation of both physiological
and pathophysiological processes, such as development, growth, tissue homeostasis,
immune responses, wound healing, inflammation and neoplasia. Alterations in cell
12
surface receptors and adhesion molecules which regulate cell-cell and cell-matrix
interactions have been implicated in these tumour processes (Albelda, 1993). There
are four major groups of cell adhesion molecules (CAMs); cadherins, integrins,
immunoglobulin superfamily, and selectins.
1.4.1.2. Tumour cell invasion
Tumour cell invasion is the active migration of neoplastic cells out of their tissue of
origin into adjacent tissues of different types. The process is completed in 3 steps.
The first step is tumour cell attachment via cell surface receptors which specifically
bind to components of the matrix such as laminin (for the basement membrane) and
fibronectin (for the stroma). The anchored tumour cell next secretes hydrolytic
enzymes (or induces host cells to secrete enzymes) which can locally degrade the
matrix (including degradation of the attachment components). Matrix lysis most
probably takes place in a highly localized region close to the tumour cell surface.
The third step is tumour cell locomotion into the region of the matrix modified by
proteolysis. An early step in locomotion is pseudopodial protrusion at the leading
edge of the migrating cell. Continued invasion of the matrix may take place by cyclic
repetition of these three steps (Liotta, 1986).
1.4.1.2.1. Attachment to ECM
The ability of tumour cells to form transient attachments is necessary for metastasis.
Metastasizing tumour cells must be able to attach to extracellular matrix components
and to other cells (of the same or different type). ECM is mainly composed of
several proteoglycans as co-receptors and proteins such as collagen, laminin, and
fibronectin. Integrins may play a critical role in the attachment of tumour cells to the
extracellular matrix (Danen et al., 1995). Collagens are the most abundant protein
constituents of ECM. Among these, type IV collagen is the major component of the
basal lamina. Laminin binds to type IV collagen and the cell membrane as a
structural component of all basement membranes, and mediates cell-matrix
interactions to promote adhesion protein involved in cell adhesion, growth,
migration, and differentiation (Choi et al., 2010).
1.4.1.2.2. Degradation of ECM
In order for cells to migrate, they must be able to degrade the protein barriers.
Degradation of basement membrane is a crucial event in invasion. Basement
13
membranes and connective tissues consist of four major groups of molecules:
collagens, elastin, glycoproteins, and proteoglycans. The quantity of each of these
differs among basement membranes of different tissues. These extracellular matrix
constituents are stabilized and organized by various protein-protein and
polysaccharide-protein interactions that can be destabilized by degradative enzymes
(Fidler, 1991), generally called proteases.
1.4.1.2.2.1. Protease: Serine protease, cathepsins & MMPs
In cancer, altered proteolysis leads to unregulated tumour growth, tissue
remodelling, inflammation, tissue invasion, and metastasis (Kessenbrock et al.,
2010). Proteolysis of extracellular matrix (ECM) and basement membrane is an
essential mechanism used by cancer cells for their invasion and metastasis. Many
proteases are capable of degrading extracellular matrix components. They are
divided into three groups: Serine proteases, Cathepsins, and Matrix metalloproteases
(MMPs).
1.4.1.2.2.1.1. Matrix Metalloproteinases (MMPs)
The matrix metalloproteinases (MMP) are a family of enzymes considered to be
primarily responsible for extracellular matrix (ECM) degradation. These enzymes
secreted in inactive proenzymatic forms, are zinc-dependent, and can be subdivided
on the basis of preferential extracellular matrix substrate (Kessenbrock et al., 2010).
In particular, the MMPs include the only enzymes known to be capable of degrading
fibrillar collagen (Curran and Murray, 2000). Matrix metalloproteinases are secreted
as proenzymes that require extracellular activation. They are divided into five
general classes: (1) interstitial collagenases, (2) stromelysins, (3) gelatinases (type IV
collagenases), (4) matrilysins and (5) Membrane-type MMPs.
MMP-2 and MMP-9 are the most important gelatinases involved in the
basement membrane collagen IV degradation. MMP-2 (72 kDa) also called
gelatinase A, constitutively expressed by most cells including endothelial and
epithelial cells. The 92 kDa MMP-9 (gelatinase B) is produced by inflammatory
cells, including blood neutrophils and tissue macrophages, as well as by stimulated
connective tissue cells. MMP-2 is secreted as an inactive pro-form; when it is
converted to a 62 kDa active form it can degrade collagen types IV and V, laminin
and elastin. Membrane-type matrix metalloproteinase (MT1-MMP) appears to be
14
important in cellular activation of MMP-2 (Foda and Zucker, 2001). A positive
correlation between tumour progression and the expression of MMP-2 and MMP-9
in tumour tissues has been demonstrated in numerous human and animal studies.
Metastatic cells produce specialised cell-surface structures called invadopodia,
which utilize proteases to degrade ECM components such as fibronectin, laminin,
type I and IV collagens etc.
1.4.1.2.2.1.1.1. Regulation of MMPs
MMP activity is regulated at three levels: transcription, proteolytic activation of the
zymogen and inhibition of the active enzyme. MMPs are expressed in tissues at
various stages of development but are typically absent in normal cells of the adult
organism (Stamenkovic, 2000). MMPs can be activated by various external stimuli,
including cytokines, growth factors and changes in cell–cell and cell–ECM
interactions.
MMP expression is primarily regulated at the transcriptional level, which is
regulated either positively or negatively by cytokines and growth factors such as
interleukins (IL-1, IL-4, and IL-6), transforming growth factors (EGF, HGF, and
TGF-β), or tumour necrosis factor alpha (TNF-α) (Kupai et al., 2010). MMPs are
generally expressed in very low amounts in latent forms (Pro-MMP). Generally
MMPs are kept inactive by interaction between cystein-sulphhydryl group in pro-
peptide domain and the Zn+ bound to catalytic domain. Activation requires the
proteolytic removal of pro-peptide domain and this mechanism of activation is called
“cystein switch”. Serine proteinase, plasmin has been observed to proteolytically
activate a number of latent MMPs (Kessenbrock et al., 2010).
1.4.1.2.2.1.1.2. Tissue inhibitors of metalloproteinases (TIMPs)
The proteolytic activity of MMPs is primarily controlled by its natural inhibitors,
Tissue inhibitors of metalloproteinases (TIMPs). They are consequently important
regulators of ECM turnover, tissue remodelling and cellular behaviour (Brew and
Nagase, 2010). TIMPs are small proteins of 21–28 kDa that specifically block MMP
activity by binding to the highly conserved zinc-binding site of active MMPs. There
is a balance between the levels of activated MMPs and TIMPs in normal cells and
altering this equilibrium affects the process of cellular invasion. There are currently
four known TIMP family members; TIMP-1, TIMP-2, TIMP-3 and TIMP-4. TIMP-1
15
and TIMP-2 inhibit the activity of most MMPs. TIMP-1 can form a complex with
pro-MMP-9, similarly TIMP-2 with pro-MMP-2 (Stamenkovic, 2000). Increased
TIMP-1 and TIMP-2 levels in human cancers have been associated with poor
prognoses (Foda and Zucker, 2001). Numerous studies correlate low TlMP
expression with enhanced invasive and metastatic properties in a number of murine
and human tumour cell lines. Therefore, TlMPs play a critical role in the regulation
of tissue degradation in metastatic diseases, but their use as pharmacologic inhibitors
of MMPs has been limited due to thier short half-life, when administered in vivo
(Ray and Stetler-Stevenson, 1994).
1.4.1.2.3. Migration
In order for cancer cells to spread to distant sites, they often must first migrate away
from the primary tumour as part of the invasive process. Cell migration across
extracellular matrix completed in a continuous cycle of interdependent steps. First,
the moving cell becomes polarized and elongated. 1) Protrusion of the cell
membrane forms pseudopodia that is mediated by actin polymerization, which
attaches to the ECM substrate and 2) forms focal adhesion complex (FAK) 3)
inactivated integrin subunits continually recycle along the cell surface 4) change in
integrin affinity engages the ECM and a force is generated 5) the FAK is disrupted at
the trailing edge of the cell 6) integrin complexes release (proteolytic disruption of
FAK). During this cascade, regions of the leading edge or the entire cell body
contract, thereby generating traction force that leads to the gradual forward gliding
of the cell body and its trailing edge (Gupta and Massagué, 2006). Numerous
migration factors have been identified that appear to be associated with cancer cell
migration (Oppenheimer, 2006). A series of proteins on the surface of the
pseudopodia coordinate sensing, protrusion, burrowing, and traction. Proteinases at
the pseudopodia tip may locally disrupt the extracellular matrix and permit forward
extention (Kohn and Liotta, 1995).
1.4.1.3. Intravasation
For cancer cells to spread to distant organs they need to enter either the blood or
lymphatic vasculature. Tumour cells enter into blood stream by digesting basement
membrane. The mechanistic cascade is similar to invasion. Metastatic tumour cells
first attach to the endothelial basement membrane, digest it by secreting powerful
16
proteases, and migrate between the endothelial cells to enter the bloodstream. Entry
into lymphatic vasculature is also an important route of tumour cell dissemination as
the process is simpler due to the absence of a basement membrane.
1.4.1.4. Cancer cells in the blood stream
It is true that the majority of disseminating tumour cells die rapidly in the circulation
due to nonspecific factors such as hemodynamic forces. They undergo severe
damage and a large fraction of them (over 90%) die within 24 hours after
intravasation. Lack of adhesion substrates, collision with cellular components in the
blood stream, passage through narrow capillaries, and contact with blood
components are all believed to influence the viability of circulating tumour cells
(Kawaguchi, 2005). Although most tumour cells are quickly destroyed within the
bloodstream, it appears that the greater the number of cells released by a primary
tumour, they travel in clusters, increases the possibility that at least one will survive.
Host factors such as T-cell, natural killer (NK) cell, endothelial cells, and
macrophage activity may destroy circulating tumour cells (Xie and Huang, 2003).
Tumour cells are surrounded with blood cells such as lymphocytes and platelets,
which mask the cancer cells from immune surveillance.
1.4.1.5. Extravasation
Escape of tumour cells from blood vessels into surrounding tissues is called
extravasation. The process is very similar to intravasation. First tumour cells in the
circulation attach to the endothelial cells and then onto the basement membrane.
α4β 1 integrins or tumour cell proteoglycans (carbohydrate side chains) on tumour
cells facilitate the adhesion to endothelial vascular adhesion molecules. Tumour cells
digest the basement membrane by secreting protease and move through it (Neal and
Berry, 2006).
1.4.1.6. Implantation into target organ: Specificity of metastasis
Organ-specific homing and colonization of cancer cells (Tissue tropism) are
important and interesting features of metastasis. Tissue tropism depends on both the
histology and the stage of cancer. Cancers, such as breast, metastasize to multiple
sites, most commonly lung and bone tissue and with less frequency to the liver and
the adrenal glands. Prostate cancer preferentially spreads to bone. Patients with
colorectal cancer, by contrast, often develop initial metastases in liver (Chambers et
17
al., 2002). Stephen Paget, 1989 proposed a “seed and soil” hypothesis that site
specific lodging patterns were due to the ‘dependence of the seed (the cancer cell) on
the soil (the secondary organ). Carbohydrates on the cancer-cell surface bind to a
specific receptor selectin on the endothelial cells. Different selectins recognise
different carbohydrates on the cancer cell surface. Each cancer-cell type expresses a
different set of carbohydrates on its surface, attracts to different selectin molecules.
These interactions account for the differential homing specificities of different types
of cancer cells.
1.4.1.7. Tumour angiogenesis
Angiogenesis is a biological process of endothelial cell (EC) sprouting to form new
blood vessels from existing ones. Neovascularisation is a requirement for solid
tumour growth beyond 1–2 mm in diameter (Folkman, 1990). So angiogenesis is a
crucial event in cancer metastasis. The angiogenic process is a balance between
stimulatory and inhibitory factors. A change that favours stimulation may trip an
‘angiogenic switch’ allowing the tumour to induce the formation of microvessels
from the surrounding host vasculature (Hanahan and Folkman, 1996). Tumours
promote angiogenesis by secreting or inducing the release of growth factors that
stimulate endothelial cell migration and proliferation, proteolytic activity and
capillary morphogenesis (discussed in detail in section 1.5.).
1.4.2. Metastasis suppressor genes
Metastasis suppressor genes (MSGs) do not affect the growth of the primary tumour
but significantly inhibit the steps in the metastatic cascade and reduce the formation
of secondary tumour. Many MSGs inhibit tumour cell motility and invasion in the
primary tumour. Other MSGs regulate tumour cell arrest, extravasation, survival and
growth at the secondary. Twenty-three such genes have now been described in the
literature and collectively make up the metastasis suppressor gene family.
The first metastasis suppressor, nm23 (non-metastatic clone-23), was
identified in 1988. nm23-H1 has been shown to suppress metastasis in several cancer
models such as melanoma, prostate, colon, breast and oral squamous cell
carcinomas. Studies demonstrated that transfection of the nm23-H1 gene to murine
melanoma cell lines can suppress their metastatic potential (Horak et al., 2008).
Transfection of nm23 gene into highly metastatic K-1735 melanoma cells reduced
18
their in vivo metastatic ability by 52% to 96%, with no effect on the primary tumour
size (Leone et al., 1991).
1.5. TUMOUR ANGIOGENESIS
Tumours cannot grow beyond a size of approximately 1–2mm without
neovascularization (Folkman, 1990). Angiogenesis, a crucial event in cancer
metastasis, is the sprouting of new capillaries from existing blood vessels, consists of
a complex cascade of events including endothelial cell–mediated degradation and
invasion of the extracellular matrix, endothelial cell migration, proliferation,
differentiation, basement membrane deposition, and the organization of endothelial
cords into capillary structures (Soriano et al., 2004).
Angiogenesis is mediated by several pro-angiogenic molecules released by
both tumour cells and host cells including endothelial cells, epithelial cells,
mesothelial cells and leucocytes. Among these, vascular endothelial growth factor
(VEGF or vascular permeability factor, vasculotropin), members of the fibroblast
growth factor (FGF) family, interleukin-8 (IL-8), angiogenin, angiotropin, epidermal
growth factor (EGF), fibrin, nicotinamide, platelet derived endothelial cell growth
factor (PD-ECGF), platelet derived growth factor (PDGF), transforming growth
factor- α (TGF-α), TGF-β, and tumour necrosis factor-α (TNF-α) are important
regulators (Ellis and Fidler, 1996). These pro-angiogenic factors bind to receptors on
nearby blood vessels and induce the activation, proliferation and migration of
endothelial cells towards the tumour. Stimuli for the production of pro-angiogenic
mediators include hypoxia, cytokines and growth factors, as well as mutations in
oncogenes and tumour suppressor genes (Neal and Berry, 2006). The integrin family
of cell adhesion proteins mediates cell attachment to the extracellular matrix and
promotes the survival, proliferation, and motility of ECs during angiogenesis
(Varner, 1997).
The process of angiogenesis can be completed in four steps, similar to
tumour cell invasion process: (1) proliferation of endothelial cells (2) breakdown of
the extracellular matrix (3) migration of endothelial cells and (4) tube formation and
structural reorganization. Under normal physiological conditions angiogenic
mediators establish a balance between local pro-angiogenic and antiangiogenic
functions by the receptors on the surface of endothelial cells. In response to an
19
angiogenic stimulus (injury, inflammation, hypoxia) endothelial cells (EC) become
activated, attract and bind leukocytes and blood platelets that release a multitude of
pro- and anti-angiogenic factors (Patan, 2000). The EC further loosen their contacts
with each other, their basement membrane (BM) and their supporting peri-
endothelial cells (pericytes and smooth muscle cells (SMC) leading to increased
vascular permeability and deposition of fibrin into the extra-vascular space, vessel
wall disassembly and BM degradation. The activated EC migrate on and into the
fibrin scaffold and invade the underlying extra-cellular matrix (ECM) towards the
angiogenic stimulus and proliferate. Ultimately they form a capillary lumen by
aligning. Once a new vessel has been formed, EC proliferation and migration are
inhibited and a new BM is secreted. The junctional complexes between the EC as
well as with the BM mature and peri-endothelial cells are recruited and differentiate.
Yet, sprouting is not the only way to enlarge the vascular network: larger vessels can
split longitudinally into two daughter vessels by intussusception (Patan, 2000).
Furthermore, bone-marrow derived endothelial precursor cells have been shown to
home to sites of blood vessel growth, differentiate, proliferate and form new vessels
(Rafii and Lyden, 2003). Finally, vascularisation can be augmented by enlargement
of pre-existing vessels as is seen in collateral outgrowth (Bouis et al., 2006).
1.5.1. Angiogenic switch
In pathological states such as chronic inflammation and tumour growth, there is an
imbalance between endogenous stimulator and inhibitor levels, leading to an
"angiogenic switch" (Ribatti, 2009) and has been recognized as key in promoting the
transition towards a clinically aggressive tumour and that promotes and facilitates
‘‘neo-angiogenesis’’. Pro- and anti-angiogenic factors arise from cancer cells,
stromal cells, endothelial cells, inflammatory cells, the extracellular matrix (ECM),
and blood. Importantly, the relative contribution of these factors is dependent on the
tumour type and site, and their expression changes with tumour growth, regression,
and relapse. Tumour growth is currently viewed as a phenomenon associated with
neovascularization and sustained production of angiogenic factors, studies have
emphasized that tumour angiogenesis is a process requiring a higher amount of
angiogenic factors for its induction than maintenance (Dong et al., 2007).
20
1.5.2. Angiogenic factors
More than 200 proangiogenic factors have been identified. Among those, the most
important factors include vascular endothelial growth factor (VEGF), fibroblast
growth factor (FGF), angiopoietin (Ang) and chemokines. These factors initiate
angiogenesis by modulating the migration and/or proliferation of EC and the
formation of neovasculature.
1.5.2.1. Vascular endothelial growth factor
One of the most important pro-angiogenic factor in the angiogenesis process is
vascular endothelial growth factor (VEGF), which regulates endothelial
proliferation, permeability, and survival. This glycoprotein encoded by a gene
located at chromosome 6 (6p21), is able to bind to heparin and has an 18%
homology with platelet derived growth factor (PDGF) (Ferrara and Henzel, 1989).
Under hypoxia, hypoxia inducible factor-1alpha (HIF-1α) transcription is
upregulated which then activates VEGF transcription, promoting the formation of
abnormal blood vessels (Mendoza et al., 2008). VEGF stimulates ECM degradation,
proliferation, migration, and tube formation of endothelial cells. VEGF promotes
endothelial cell proliferation via the activation of the MAPK pathway (Wong et al.,
2009). It induce survival signaling pathway by up regulating phosphatidylinositol-3’
kinase (PI3K)/Akt, ERK kinases and Bcl-2 and also by down regulating p53, p21,
p16, p27, and proapoptotic protein Bax (Nor et al., 1999, Wong et al., 2009). High
circulating levels of VEGF correlate with poor prognosis in various solid tumours
(Poon et al., 2001).
There are at least five members of the VEGF family have been described.
These are VEGF-A, -B, -C, -D, and -E, where VEGF-A is the VEGF of prime
importance for angiogenesis. Human VEGF exists in six isoforms VEGF121,
VEGF145, VEGF165, VEGF183, VEGF189, VEGF206, with the labels indicating the
number of amino acids. In vivo, only the three secreted isoforms VEGF121, VEGF145,
VEGF165 can induce angiogenesis, with VEGF165 being the predominant isoform
secreted by benign and malignant cells. VEGF189 and VEGF206 are almost totally
sequestered in the extracellular matrix, but can be released by heparinase or plasmin
proteolysis in an active soluble form. All VEGF isoforms are capable of binding to
the receptor tyrosine kinases VEGFR1 and VEGFR2 (Bremnes et al., 2006). Upon
21
binding to its receptor, VEGF initiates a cascade of signaling events that begins with
dimerization of two receptors and then autophosphorylation of each other by the
tyrosine kinase domain to form the active receptors. This is followed by activation of
numerous downstream proteins, including phospholipase C-gamma/protein kinase C
(PKC), Ras pathway members, phosphatidylinositol-3 kinase (PI3K), mitogen-
activated protein kinase (MAPK), and others to manifest end point function, such as
an increase in vascular permeability, cell survival and proliferation, and migration.
Among the many transcription factors, Sp1, HIF-1, Stat3, and AP-1 appear to be the
key factors in regulation of VEGF expression (Keping et al., 2004).
1.5.2.1.1. Vascular endothelial growth factor receptor (VEGFR)
Different VEGFs bind to receptors which exhibit tyrosine kinase (TK) activity. The
specific effect of the VEGF on the endothelial cells is primarily regulated by two
receptor tyrosine kinases (RTKs) of the VEGF family: VEGFR1, also named Flt-1
(Shibuya et al., 1990; deVries et al., 1992); VEGFR2, also named Flk- 1 or KDR
(Terman et al., 1992; Millauer et al., 1993; Quinn et al., 1993). VEGFR1 and
VEGFR2 have different signal transduction functions as VEGFR2 is the major
mediator of the mitogenic, angiogenic, and permeability-enhancing effect of VEGF,
while VEGFR1 is primarily involved in vessel differentiation/ architectural
organisation, haematopoiesis, induction of MMPs, and do not possess mitogenic
activities (Ferrara, 2004).
1.5.2.2. Basic fibroblast growth factor (bFGF)
bFGF, a soluble heparin binding polypeptide, is commonly found in malignant
tumours. bFGF has a mitogenic effect on endothelial cells, and is a potent inducer of
angiogenesis. The paracrine production of bFGF in tumour cells is associated with
the angiogenic switch described above (Kandel et al., 1991). Beyond its independent
effect on endothelial cells, bFGF also works synergistically with VEGF in inducing
angiogenesis (Asahara et al., 1995). Furthermore, monoclonal antibodies against
human bFGF inhibit tumour growth (Hori et al., 1991).
1.5.2.3. Angiopoietins
Angiopoietins are another family of endothelial cell-specific molecules that play an
important role in vessel maintenance, growth and stabilization (Yancopoulos et al.,
1998). There are four types of angiopoietins known: Ang-1, -2, -3 and -4. Ang-1 acts
22
as an agonist promoting vessel stabilization in a paracrinal manner, whereas Ang-2 is
an autocrine antagonist inducing vascular destabilization at high concentrations.
Ang-2 has been found to be dramatically increased during vascular remodelling and
is implicated in tumour-associated angiogenesis and tumour progression (Thomas et
al., 2009).
1.5.2.4. Platelet-derived endothelial cell growth factor (PD-ECGF)
The origin for its name is that PD-ECGF was first isolated from platelets (Miyazono
et al., 1987). PD-ECGF is expressed by a wide variety of tumours (Takebayashi et
al., 1996), as well as by normal cells like macrophages, stromal cells, and glial cells
(Griffiths and Stratford, 1997; Fox et al., 1995). As for VEGF, its production in
tumour cells has been associated with hypoxia in the tumour environment. PD-
ECGF increases DNA synthesis, endothelial cell migration in vitro, tumour growth
and angiogenesis in vivo (Moghaddam et al., 1995). PD-ECGF expression in both
tumour cells and stromal fibroblasts correlates with angiogenesis (Koukourakis et al.,
1998). The mechanism by which PD-ECGF promotes angiogenesis involves
stimulation of endothelial cell migration (Moghaddam et al., 1995).
1.5.2.5. Chemokines
Chemokines are known to possess pro- or anti- angiogenic activities. In more details,
CXC chemokines with glutamic acid–leucine–arginine motif (ELR+), such as
interleukin-8 (IL-8), neutrophil-activating protein-2 (NAP-2), granulocyte
chemotactic protein-2 (GCP-2), epithelial- derived neutrophil-activating protein-78
(ENA-78), growth related protein (GRO)-α, -β and -γ, induce endothelial cell
migration and proliferation as potent angiogenic factors (Makrilia et al., 2009).
1.5.2.6. Transforming growth factor-β
The transforming growth factor-β (TGF-β) is thought to have both pro- and anti-
angiogenic properties. Low TGF-β levels contribute to the angiogenic switch, by up-
regulating angiogenic factors and proteinases. On the other hand, high TGF-β levels
inhibit endothelial cell growth, stimulate smooth muscle cells differentiation,
recruitment and promote basement membrane reformation (Carmeliet, 2003). In
cancer cells, there are multiple mutations in the TGF-β signaling pathway. Both
cancer cells and the surrounding stromal cells (fibroblasts) proliferate, while TGF-β
production is also increased, affecting the surrounding stromal cells, immune cells,
23
endothelial cells and smooth muscle cells (Blobe et al., 2000). More specifically,
during the initial stages of tumourigenesis, TGF-β inhibits tumour growth and
development by inhibiting cell proliferation and by inducing apoptosis. In later
stages, tumour cells become resistant to the tumour suppressor activity of TGF-β,
TGF-β takes on a pro-oncogenic role (Pardali et al., 2009).
1.5.2.7. Tumour Hypoxia
Tumour cannot grow beyond 1-2 mm without proper blood supply (Wachsberger et
al., 2003). Hypoxia is an important pathophysiological feature in the
microenvironment of solid tumours, which is caused by the imbalance between
oxygen supply and consumption (Peng and Chen, 2010). Oxygen plays an important
role in the generation of free radicals by ionizing radiation and subsequent radiation
induced free radical mediated cancer cell damage. In the absence of oxygen, more
radiation damage is repaired; thereby the effect of radiation is compromised (Peng
and Chen, 2010). Radiation therapy therefore rescues metastatic radiation resistant
hypoxic cells from dying and hence increased the probability of hypoxia induced
metastasis. Tumour hypoxia is an environmental stimulus that plays a key role in the
development and cancer progression, and is one of the major causes of treatment
failure in radiotherapy and chemotherapy in cancer patients. The hypoxic response is
mainly mediated by hypoxia inducible factor-1 (HIF-1) composed of HIF-1α and
HIF-1β, which becomes active under low oxygen condition. HIF-1 is generally
regarded as the master regulator of the hypoxia response and is known to regulate
several genes involved in regulation of metabolism, tumour angiogenesis and
metastasis (Yang and Wu, 2008; Semenza, 2003). In normoxia, HIF-1α is
hydroxylated and targeted for ubiquitination and proteasomal degradation (Denko,
2008). In hypoxic conditions it translocates to the nucleus where it dimerises with
HIF-1β and binds to hypoxia responsive elements and activates transcription (Azam
et al., 2010). Expression of HIF-1α is upregulated in many human cancers and is
associated with treatment failure (Koukourakis et al., 2006, Bos et al., 2003). Tissue
hypoxia can induce a number of angiogenic factors that promote angiogenesis, such
as VEGF, IL-8, angiogenin, FGF, and PDGF. HIF-1α can stimulate the expression of
vascular endothelial growth factor (VEGF) (Tsiokas et al., 1995). VEGF plays a
major role in physiological blood vessel formation and pathological angiogenesis in
tumour growth (Ellis and Hicklin, 2008). Hypoxia-induced upregulation of VEGF as
24
well as angiogenic cytokines and downmodulation of antiangiogenic factors by
oncogenes or tumour suppressor genes are currently thought to be the basic
molecular mechanisms responsible for angiogenesis in solid tumours (Moehler et al.,
2003). Several studies demonstrated that many antiangiogenic drugs could overcome
tumour hypoxia and improve tumour response to radiation therapy (Wachsberger et
al., 2003).
1.5.3. Naturally occurring angiogenic inhibitors
1.5.3.1. Thrombospondin-1
A 140 kDa fragment of thrombospondin-1 (TSP-1) was one of the first natural
angiogenic inhibitors to be described (Rastineiad et al., 1989). TSP-1 is an inhibitor
of tumour growth and metastasis in a number of animal models. Its expression is
inversely correlated with angiogenic activity. For example, TSP-1 is down-regulated
during tumourigenesis while angiogenesis activity is elevated (Rastineiad et al.,
1989). In subsequent studies of fibroblasts cultured from patients with Li-Fraumeni
disease, it was shown that TSP-1 is regulated by the p53 tumour suppressor gene
(Dameron et al., 1994). Loss of p53 results in suppression of TSP-1 and a
concomitant increase in angiogenic activity.
1.5.3.2. Interferon
The anti endothelial activity of interferon has been known for some time. One
potential mechanism of interferon action may be to block the production or efficacy
of angiogenic factors produced by tumour cells (Singh et al., 1995). Some vascular
tumours are more sensitive to the inhibitory activity of interferon. Hemangiomas,
large benign tumours comprised predominantly of endothelial cells, are particularly
sensitive to treatment with interferon. Treatment with α-interferon is one of the first
clinically successful treatment protocols for patients with proliferating hemangiomas
(Zetter, 1998).
1.5.3.4. Metalloproteinase Inhibitors
Naturally occurring MMP inhibitors, known as TIMPs (tissue inhibitors of
metalloproteinases), have been found in a variety of cells and tissues. All members
of the TIMP family inhibit angiogenesis (Johnson et al., 1994). In addition, a
naturally occurring angiogenic inhibitor isolated from cartilage has TIMP-like
domains. TIMPs also inhibit tumour growth and metastasis (Imren et al., 1996). The
25
mechanism whereby TIMPs inhibit angiogenesis and metastasis would appear to be
their ability to suppress matrix degradation (Zetter, 1998).
1.5.4. Tumour-derived inhibitors
1.5.4.1. Angiostatin
The most potent and specific inhibitors of angiogenesis are the products of
proteolytic cleavage of larger proteins. Angiostatin, a 38-kDa fragment of
plasminogen, was first characterized by O’Reilly et al. (1994) as a circulating
antiangiogenic factor discovered in a murine Lewis lung carcinoma model. In several
murine tumour models, systemic administration of angiostatin resulted in dormancy
of metastasis (O’Reilly et al., 1996).
1.5.4.2. Endostatin
Endostatin is a 20 kDa carboxy-terminal fragment of collagen XVIII, purified from
the conditioned media of hemangioendothelioma cells (O’Reilly et al., 1997). Like
angiostatin, endostatin is a cryptic angiogenesis inhibitor released from a larger
parent molecule that itself is not anti-angiogenic (Hanahan and Folkman, 1996). One
is that endostatin does not induce drug resistance as do conventional chemotherapy
and radiation. In addition, in mouse tumour models, repeated cycles of administering
and withdrawing systemic endostatin results in prolonged tumour dormancy without
further treatment, suggesting that endostatin could completely suppress a tumour
rather than just inhibit it transiently (Boehm et al., 1997).
1.5.5. Pharmacologic agents that inhibit angiogenesis
1.5.5.1. TNP-470
The synthetic fumagillin analogue, O-(chloro-acetylcarbamoyl) fumagillol (TNP-
470), was 50-fold more potent than fumagillin and generally well tolerated. The anti-
angiogenic effect of TNP-470 appears to be mediated by the perturbation of
endothelial cell cycle regulators or promotion of thrombospondin-1 production (Abe
et al., 1994). TNP-470 inhibited androgen-independent PC-3 xenografts in nude
mice by 96%, with an additive antitumour effect when combined with cisplatin
treatment. Combined therapy with docetaxel and TNP-470 showed a synergistic
effect against murine subcutaneous and orthotopic PC-3 tumours (Muramaki et al.,
2005).
26
1.5.5.2. Thalidomide
An anti-angiogenic property of thalidomide was first described by D’Amato et al.
(1994) against the neovascularization of a bFGF-stimulated rabbit cornea model. In
human beings, thalidomide is antiangiogenic only when metabolized to its active
product (Bauer et al., 1998). Figg et al. (2001a) tested the clinical use of thalidomide
in men with metastatic prostate cancer who had multiple therapies failure. The data
from these clinical trials are very promising. Currently, thalidomide analogues are
being developed with higher anti-angiogenic activities and wider therapeutic
windows (Capitosti et al., 2004; Ng et al., 2004).
1.5.5.3. CAI
Carboxyamidotriazole (CAI) is a calcium channel inhibitor that blocks tumour cell
migration, proliferation and has antiangiogenic activity. CAI retards metastasis in
experimental animals and has completed phase I clinical trials in cancer patients.
Published results from these trials showed disease stabilization in 49% of the
patients who had disease progression before starting CAI treatment (Kohn et al.,
1996). Further evaluation of this intriguing multimodal antitumour agent is currently
underway.
1.5.5.4. Troponin
Troponin I (TnI) a 22 kDa angiogenic inhibitor isolated from cartilage (Moses et al.,
1999), inhibits FGF- and VEGF-driven capillary endothelial cell proliferation in
vitro and neovascularization in chorioallantoic membrane (CAM) and cornea
models. TnI delivered systemically, significantly inhibited lung metastases of murine
B16-BL6 melanoma, a very aggressive variant of B16- F10 melanoma.
1.6. APOPTOSIS
Apoptosis is the most common and well-defined form of programmed cell death. It
is a physiological cell suicide that is essential for the maintenance of homeostasis in
embryonic, fetal and adult tissues (Kerr et al., 1972; Okada and Mak, 2004). Defects
in apoptotic mechanisms play important roles in tumour pathogenesis, allowing
neoplastic cells to survive beyond their normally intended life spans, subverting the
need for exogenous survival factors, providing protection from hypoxia and
oxidative stress as tumour mass expands, and allow time for accumulative genetic
alterations that deregulate cell proliferation, interfere with differentiation, promote
27
angiogenesis, and increase cell motility and invasiveness during tumour progression
(Reed, 1999). In fact, apoptosis defects are recognized as an important complement
to protooncogene activation, as many deregulated oncoproteins that drive cell
division and apoptosis (e.g., Myc, E1a, Cyclin-D1) (Green and Evan, 2002).
Similarly, defects in DNA repair and chromosome segregation normally trigger cell
suicide as a defense mechanism for eradicating genetically unstable cells, and thus
apoptosis defects permit survival of genetically unstable cells, providing
opportunities for selection of progressively aggressive clones (Ionov et al., 2000).
Apoptosis defects also facilitate metastasis by allowing epithelial cells to survive in a
suspended state, without attachment to extracellular matrix (Frisch and Screaton,
2001). They also promote resistance to the immune system, in as much as many of
the weapons cytolytic T cells (CTLs) and natural killer (NK) cells use for attacking
tumours depend on integrity of the apoptosis machinery (Tschopp et al., 1999).
Finally, cancer-associated defects in apoptosis play a role in chemoresistance and
radioresistance, increasing the threshold for cell death and thereby requiring higher
doses for tumour killing (Makin and Hickman, 2000). Thus, defective apoptosis
regulation is a fundamental aspect of cancer biology.
1.6.1. Morphology of Apoptosis
During the early process of apoptosis, cell shrinkage and pyknosis are visible by
light microscopy (Kerr et al., 1972). With cell shrinkage, the cells are smaller in size,
the cytoplasm is dense and the organelles are more tightly packed. Pyknosis is the
result of chromatin condensation and this is the most characteristic feature of
apoptosis. Extensive plasma membrane blebbing occurs followed by karyorrhexis
and separation of cell fragments into apoptotic bodies during a process called
“budding.” Apoptotic bodies consist of cytoplasm with tightly packed organelles
with or without a nuclear fragment (Elmore, 2007).
1.6.2. Apoptotic Pathways
The mechanisms of apoptosis are highly complex and sophisticated, involving an
energy-dependent cascade of molecular events. Apoptosis can be triggered in a cell
through two main apoptotic pathways either the extrinsic pathway or the intrinsic
pathway. There is an additional pathway that involves T-cell mediated cytotoxicity
28
and perforin-granzyme-dependent killing of the cell. Cancer may arise from the
dysfunction in the apoptotic pathway.
1.6.2.1. The Extrinsic Pathway
In the extrinsic pathway, signal molecules known as ligands, which are released by
other cells, bind to transmembrane death receptors on the target cell to induce
apoptosis. The death receptors are mainly members of the tumour necrosis factor
(TNF) receptor gene superfamily (Locksley et al., 2001). They share similar
cysteine-rich extracellular domains and have a cytoplasmic domain of about 80
amino acids called the “death domain” (Ashkenazi and Dixit, 1998). This death
domain plays a critical role in transmitting the death signal from the cell surface to
the intracellular signaling pathways. To date, the best-characterized ligands and
corresponding death receptors include FasL/FasR, TNF-α/TNFR1, Apo3L/DR3,
Apo2L/DR4 and Apo2L/DR5 (Elmore, 2007). For example, the immune system’s
natural killer cells possess the Fas ligand (FasL) on their surface (Csipo et al., 1998).
The binding of the FasL to Fas receptors (a death receptor) on the target cell will
trigger multiple receptors to aggregate together onto the surface of the target cell.
The aggregation of these receptors recruits an adaptor protein known as Fas-
associated death domain protein (FADD) on the cytoplasmic side of the receptors.
FADD, in turn, recruits caspase-8, an initiator protein, to form the death-inducing
signal complex (DISC). Through the recruitment of caspase-8 to DISC, caspase-8
will be activated and it is then able to directly activate caspase-3, an effector protein,
to initiate degradation of the cell. Active caspase-8 can also cleave BID protein to
tBID, which acts as a signal on the membrane of mitochondria to facilitate the
release of cytochrome c in the intrinsic pathway (Adrain et al., 2002).
1.6.2.2. The Intrinsic Pathway
The intrinsic pathway is triggered by cellular stress, specifically mitochondrial stress
caused by factors such as DNA damage and heat shock (Adrain et al., 2002). Upon
receiving the stress signal, the proapoptotic proteins in the cytoplasm, BAX and
BID, bind to the outer membrane of the mitochondria to signal the release of the
internal content. However, the signal of BAX and BID is not enough to trigger a full
release. BAK, another proapoptotic protein that resides within the mitochondria, is
also needed to fully promote the release of cytochrome c and the intramembrane
29
content from the mitochondria (Hague and Paraskeva, 2004). Following the release,
cytochrome c forms a complex in the cytoplasm with adenosine triphosphate (ATP),
an energy molecule, and Apaf-1, an enzyme. Following its formation, the complex
will activate caspase-9, an initiator protein. In return, the activated caspase-9 works
together with the complex of cytochrome c, ATP and Apaf-1 to form an apoptosome,
which in turn activates caspase-3, the effector protein that initiates degradation.
Besides the release of cytochrome c from the intramembrane space, the
intramembrane content released also contains apoptosis inducing factor (AIF) to
facilitate DNA fragmentation, and Smac/Diablo proteins to inhibit the inhibitor of
apoptosis (IAP) (Hague and Paraskeva, 2004).
1.6.2.3. Perforin/granzyme Pathway
T-cell mediated cytotoxicity is a variant of type IV hypersensitivity where sensitized
CD8+ cells kill antigen-bearing cells. These cytotoxic T lymphocytes (CTLs) are able
to kill target cells via the extrinsic pathway also able to exert their cytotoxic effects
on tumour cells or virus-infected cells via a novel pathway that involves secretion of
the transmembrane pore-forming molecule perforin with a subsequent exophytic
release of cytoplasmic granules through the pore and into the target cell (Trapani and
Smyth, 2002). The serine proteases granzyme A and granzyme B are the most
important component within the granules. Granzyme B cleaves proteins at aspartate
residues and activates pro-caspase-10 and also can cleave factors like ICAD
(Inhibitor of Caspase Activated DNase) (Sakahira et al., 1998).
1.6.2.4. Execution Pathway
The extrinsic and intrinsic pathways both end at the point of the execution phase,
considered the final pathway of apoptosis. It is the activation of the execution
caspases that begins this phase of apoptosis. Execution caspases activate cytoplasmic
endonuclease, which degrades nuclear material, and proteases that degrade the
nuclear and cytoskeletal proteins. Caspase-3, caspase-6, and caspase-7 function as
effector or “executioner” caspases, cleaving various substrates including
cytokeratins, PARP, the plasma membrane cytoskeletal protein alpha fodrin, the
nuclear protein NuMA and others, that ultimately cause the morphological and
biochemical changes seen in apoptotic cells (Slee et al., 2001). Caspase-3 is
considered to be the most important of the executioner caspases and is activated by
30
any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3
specifically activates the endonuclease CAD. In proliferating cells, CAD is
complexed with its inhibitor, ICAD. In apoptotic cells, activated caspase-3 cleaves
ICAD to release CAD (Sakahira et al., 1998). CAD then degrades chromosomal
DNA within the nuclei and causes chromatin condensation. Caspase-3 also induces
cytoskeletal reorganization and disintegration of the cell into apoptotic bodies.
Gelsolin, an actin binding protein, has been identified as one of the key substrates of
activated caspase-3 (Elmore, 2007).
1.6.3. Bcl-2 family
Mitochondrial outer membrane permeability is a key process involved in intrinsic
apoptotic pathway. Regulator of this process is the Bcl-2 (B-cell lymphoma 2/ family
of proteins) (Cory and Adams, 2005; Pelengaris and Khan, 2006; Roussel, 2006).
The family comprises both antiapoptotic or prosurvival members (with inhibitory
effects on apoptosis) and proapoptotic members (which block the effects of
inhibitors) (Hockenbery et al., 1990; Lowe et al., 2004). The balance between them
determines whether or not a cell commits apoptosis (Pelengaris and Khan, 2006).
The Bcl-2 family members can be subdivided according to their structure and
function. Antiapoptotic members may be divided into two subclasses: Bcl-2, Bcl-XL
and Bcl-w and another comprising Mcl-1 and A1 (Pelengaris and Khan, 2006).
Proapoptotic members are subdivided to BAX subfamily (which includes BAX,
BAK and BOK) and BH3-only subfamily (which includes BID, BIM, BAD, BIK,
BMF, PUMA, NOXA and HRK). BH3-only proteins are kept inactive in healthy
cells.
1.6.4. Caspases – the executioners of apoptosis
Caspases (Cysteine aspartic acid proteases) are highly selective cysteine proteases
that control all steps of apoptosis. The family of caspases includes 14 members
(Pelengaris and Khan, 2006). Caspases are present in every cell as inactive
precursors called procaspases (Green and Kroemer, 2004). Caspases can be grouped
into two types: “initiator” caspases and “effector” or “executioner” caspases. The
initiator caspases (e.g. caspase 8, caspase 9 and others) act by activating other
procaspases which become effector caspases (e.g. procaspase 3 and 7 can be
activated by caspases 8, 9 and others) (Green and Kroemer, 2004). In extrinsic
31
apoptotic pathway caspase 8 activate the executioner caspases 3 and 7 (Green and
Kroemer, 2004), where as caspase-9 activate the executioner caspases in intrinsic
pathway. The activity of caspases is essential for both extrinsic and intrinsic
pathways (Pelengaris and Khan, 2006; Roussel, 2006).
1.6.5. Role of p53
The tumour suppressor gene p53 has been termed the ‘guardian of the genome’,
given its essential role in surveillance of DNA damage, regulation of the cell cycle,
and regulatory role in apoptosis. The wild-type p53 gene is essential for regulation of
cell growth, and it is easy to understand that in a general sense, loss of p53 function
may be involved in the early steps of tumour formation through the survival of cells
with genetic mutations. Current evidence suggests that p53 functions to detect DNA
damage and subsequently arrest cells in the Gl phase of the cell cycle to allow for
repair; however, if the damage cannot be repaired, then apoptotic cell death is
triggered (Oren, 1994). The signal(s) that determine irreparable DNA damage is
unclear, as is the stimulus to undergo apoptotic cell death. There seems to be some
evidence, that the relative cellular content of p53 determines the response following
detection of genetic damage; when p53 content is low to moderate, cells undergo cell
cycle arrest and attempted DNA repair, though when p53 content is high, cells
progress to apoptosis (Ronen et al., 1996). As noted above, loss of cell cycle control
probably serves as an additional stimulus of p53-mediated apoptosis. A dramatic
illustration of the importance of this response can be found in viral strategies to
suppress it: adenovirus, papilloma virus, and others express proteins that sequester
p53 and prevent it from promoting apoptosis subsequent to viral infection.
A number of human cancers, including lung, colon, breast and pancreas have
been shown to harbour mutations in the p53 gene, and furthermore, the mutations
seem to occur early in the neoplastic process (Sinicrope et al., 1995). p53 appears to
mediate death through a variety of mechanisms. There is some information on the
direct interplay of p53 and apoptosis. These include down-regulation of the anti-
apoptotic genes Bcl-2, Map4 and survivin, and up-regulation of proapoptotic genes
Bax, IGF-BP3, DR5, Fas, Apaf 1 and various other apoptosome components (Slee et
al., 2004). p53 can also up-regulate PTEN, a negative regulator of the PI3K/AKT
survival pathway (Fridman and Lowe, 2003). There is evidence for the translocation
of stress-induced p53 directly to the mitochondria to induce apoptosis via Bcl-2/Bcl-
32
xl-mediated cytochrome c release, demonstrating multiple roles for p53 in mediating
cell death (Mihara et al., 2003). Given this information, p53 is an attractive target for
gene therapy as restoration of function seems to re-establish the ability to undergo
apoptosis.
1.7. NF-κB
Nuclear factor- κB (NF-κB) belonging to Rel family of proteins consists of five
members, namely: p65 (RelA), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-
κB2), and exist as homo-or heterodimers bound to the I-κB family of proteins. These
proteins contain 300 amino acids that share homology with the v-Rel oncoprotein,
known as the Rel homology domain (RHD) that is responsible for DNA binding,
dimerization to other NF-κB subunits, and nuclear translocation (Bakkar and
Guttridge, 2010). Three major forms of IκB have been identified, IκBα, IκBβ, and
IκBγ, which have been generally shown to localize NF-κB proteins in the cytoplasm.
An unusual member of the IκB family is Bcl-3, which interacts with p50 and p52
subunits of NF-κB to regulate their activity. For example, association of Bcl-3 with
the p52 subunit strongly enhances the potential of p52 to activate transcription of an
NF-κB-dependent reporter. In most cell types, NF-κB complexes are predominantly
cytoplasmic and thus transcriptionally inactive until a cell is activated by a relevant
stimulus (Baldwin, 1996; Ghosh et al., 1998). In response to pro-inflammatory
cytokines such as tumour necrosis factor (TNF) and interleukin (IL)-1, bacterial
lipopolysaccharide (LPS), or a variety of other stimuli, IκBα and IκBβ are
phosphorylated on two serine residues located within the N-terminal portion of the
peptides. This phosphorylation of IκB results in ubiquitination and subsequent
degradation by the 26S proteasome. Degradation of the IκB proteins results in the
liberation of NF-κB allowing translocation to the nucleus, where it can regulate the
expression of specific genes typically involved in immune and inflammatory
responses and in cell growth control (Baldwin, 1996; Ghosh et al., 1998; Orlowski
and Baldwin, 2002).
NF-κB has been shown to play a role in the transactivation of over 150 genes
(Pahl, 1999). These genes are involved in the regulation of fundamental processes
such as the immune response, cell adhesion, physical stress, oxidative stress, and cell
survival. However, in certain cell types and in response to certain stimuli, NF-κB has
also been shown to transactivate pro-apoptotic genes (Kucharczak et al., 2003). The
33
mechanism by which NF-κB contributes to neoplastic transformation is partly due to
its protective role against apoptosis through transcriptional upregulation of anti-
apoptotic target genes (Kucharczak et al., 2003). NF-κB pro-survival target genes
include members of the Bcl-2 family of genes Bcl-xL, Bfl1/A1 and Nr13, as well as
the cellular inhibitors of apoptosis, H-IAP1, H-IAP2, and XIAP (Kucharczak et al.,
2003). Furthermore, the two NF-κB target genes XIAP and GADD45b/MyD118
have been implicated in the inhibition of TNF-α-induced apoptosis through
downregulation of JNK activity (Kucharczak et al., 2003).
1.7.1. NF-κB-activation pathways
Three distinct NF-κB-activating pathways have emerged, and all of them rely on
sequentially activated kinases. The first pathway-the classical pathway-is triggered
by pro-inflammatory cytokines such as tumour necrosis factor (TNF)-a and leads to
the sequential recruitment of various adaptors including TNF-receptor associated
death domain protein (TRADD), receptor interacting protein (RIP) and TNF-
receptor-associated factor 2 (TRAF2) to the cytoplasmic membrane. This is followed
by the recruitment and activation of the classical IκB-kinase (IKK) complex, which
includes the scaffold protein NF-κB essential modulator (NEMO; also named IKKγ),
IKKα and IKKβ kinases. Once activated, the IKK complex phosphorylates IκBα on
Ser32 and Ser36, and is subsequently ubiquitinated and degraded via the proteasome
pathway (Viatour et al., 2005).
The second pathway - the alternative pathway - is NEMO-independent and is
triggered by cytokines such as lymphotoxin b, B-cell activating factor (BAF) or the
CD40 ligand and by viruses such as human T-cell leukaemia virus and the Epstein–
Barr virus. This signaling pathway relies on the recruitment of TRAF proteins to the
membrane and on the NF-κB-inducing kinase (NIK), which activates an IKKα
homodimer – the substrate of which is the ankyrin-containing and inhibitory
molecule p100. Once phosphorylated by IKKα on specific serine residues located in
both the N- and C-terminal regions, p100 is ubiquitinated and cleaved to generate the
NF-κB protein p52, which moves as heterodimer with RelB into the nucleus. In both
cases, phosphorylation of the inhibitory molecules is essential for the activation of
NF-κB, and this has been demonstrated by the inability of cells expressing an IκBα
34
mutant that does not become phosphorylated to activate NF-κB (Viatour et al.,
2005).
The third signaling pathway is classified as a typical because it is
independent of IKK, still requires the proteasome and is triggered by DNA damage
such as UV or doxorubicin. UV radiation induces IκBα degradation via the
proteasome, but the targeted serine residues are located within a C-terminal cluster,
which is recognized by the p38-activated casein kinase 2 (CK2). Oxidative stress
also leads to NF-κB activation via IκBα tyrosine phosphorylation. The N-terminal
Tyr42 residue is crucial for this pathway, and the Syk protein tyrosine kinase seems
to be required for H2O2- mediated NF-κB activation. All of these phosphorylation
events are signal-induced. However, IκBa is also constitutively phosphorylated on
Ser293 within its C-terminal Pro-Glu-Ser-Thr sequence by CK2, and this
phosphorylation is required for rapid proteolysis of IκBα (Viatour et al., 2005).
1.7.2. NF-κB and cancer
NF-κB can modulate the transcriptional activation of genes associated with cell
proliferation, angiogenesis, metastasis, tumour promotion, inflammation and
suppression of apoptosis. Aberrant NF-κB regulation has been observed in many
cancers (Luqman and Pezzuto, 2010). Tumour cell proliferation can be blocked by
regulating NF-κB pathway, which makes tumour cells more sensitive to antitumour
agents (Karin, 2006). Studies demonstrate the NF-κB mediated activation of
transcription factor, hypoxia-inducible factor-1α, which regulates the expression of
genes such as VEGF and COX-2 (Rius et al., 2008). NF-κB is a direct modulator for
HIF-1α (Uden et al., 2008). NF-κB signaling pathway can be activated by several
pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6 and tumour promoting
genes like VEGF, and COX-2 (Kawai and Akira, 2007; Hagemann et al., 2009;
Karin and Greten, 2005). Cellular stresses such as ionizing radiation and
chemotherapeutic agents also activate NF-κB (Sethi and Aggarwal, 2006). NF-κB
can regulate the activation of cytokines, adhesion molecules, angiogenic factors, and
MMPs that all have been associated with tumour progression and metastasis
(Coussens and Werb, 2002). Several studies demonstrate the positive correlation
between NF-κB and tumour metastasis (Marcello and Lakita, 2005). Thus NF-κB is
an ideal target for anticancer drug development.
35
1.8. CURRENT THERAPIES
The conventional approaches for cancer treatment advocate either surgical removal
or toxic treatments such as chemotherapy or radiation and have proven to be
potentially helpful in cancer control. Surgical removal of tumour is often used if the
cancer is only in one area of the body and has not spread. Radiation therapy and
chemotherapy are major conventional treatment modalities. In radiation therapy high
dose of X-rays damage the DNA of the cancerous tissue. Chemotherapeutic agents
destroy the cancer cells either directly or by inducing apoptosis. Some drugs halt cell
division thereby preventing cancer cell proliferation. Immunomodulators are used to
stimulate the body’s defense mechanisms that effectively fight against cancer. In
gene therapy cancer cells are genetically modified in such a way that immune system
can easily recognize and attack them. Some cancers depend on hormones to grow, in
such case cancer growth is inhibited by regulating hormone production (Hormone
therapy). Cancer vaccines are developed against tumour associated antigens
(proteins). The immune system prepared to fight these antigens and go after these
cells.
1.9. IMMUNOMODULATION BY NATURAL PRODUCTS
Immunomodulators are substances, which modify the activity of the immune system.
They have biphasic effects such that some tend to stimulate immune system which
are low while inhibit host defense parameters which are normal or already activated
(Stites et al., 1980). Immunomodulatory agents that are free from side effects and
which can be administered for long duration to obtain a continuous immune
activation are highly desirable for the prevention of diseases. They can enhance or
inhibit immunological responsiveness of an organism by interfering with its
regulatory mechanisms. Immunomodulators can regulate the cytokine production
such as tumour necrosis factor, interleukins and interferons and these cytokines may,
in turn activate T-cells or NK cells. Plant and plant products have been the basis of
treatment of human diseases since time immemorial. There are few plants reported
with known immunomodulatory activity. Viscum album a semiparasitic plant has
shown to stimulate both humoral as well as cell mediated immune response (Kuttan
and Kuttan, 1992). Similarly an extract from the plant Withania somnifera has
shown to stimulate the immune system (Davis and Kuttan, 2000a), reduce
leukocytopenia during chemotherapy (Davis and Kuttan, 2000b) and radiation
36
therapy (Kuttan, 1993) and inhibit urotoxicity induced by chemotherapeutic drug
cyclophosphamide (Davis and Kuttan, 2000b). Tinospora cordifolia which is widely
used in Indian system of medicine has been reported for its immunomodulatory and
antitumour activities (Mathew and Kuttan, 1999). Curcumin which is present in plant
Curcuma longa has shown to stimulate the immune system in animals (Antony et al.,
1999). It has also been reported to reduce the leukocytopenia in radiation
(Soudhamini and Kuttan, 1991) and chemotherapeutic drug treated animals
(Thressiamma et al., 1985). Punarnavine, an alkaloid present in the plant Boerhaavia
diffusa Linn has shown to stimulate the immune system in animals (Manu and
Kuttan, 2009). Many of the clinically used antineoplastic drugs such as
camptothecin, taxol, vincristine, vinblastine are plant derived products and several
clinical trials on the use of nutritional supplements and phytochemicals to prevent
cancer are going on now. Terpenoids, an important class of chemotherapeutic agent
is shown to enhance the immune system (Raphel and Kuttan, 2003) experimentally.
Natural products thus offer themselves in drug development and therapy against
most life threatening diseases such as cancer.
1.10. CHEMOPREVENTION BY NATURAL PRODUCTS
Cancer chemoprevention, a term coined by Sporn for the protective effects of
retinoids (Sporn and Newton, 1979), is defined today as the blocking or suppressing
of the carcinogenic process by one or several compounds (Wattenberg, 1985).
Several natural products and dietary components have been shown to function as
cancer chemopreventive agents. These natural products may disrupt many signaling
pathways, including transduction of cell surface (epidermal growth factor) or nuclear
(estrogen) receptors via inhibition of their associated tyrosine kinase activities that
regulate mitogenic signaling cascades. Alternatively, cytoprotective signal
transduction pathways may be activated in a concentration and time dependent
manner. People, who have diet consisting of fruits and green-yellow vegetables, have
lower risk of many kinds of cancer (Block et al., 1992).
Natural products used in folk and traditional medicine have been the
mainstay of modern cancer medicine. It begins with the use of vincristine and
vinblastine from Catharanthus roseus and in late 1960s to cure Hodgkins lymphoma
(Mann, 2002). Development of the structurally and mechanistically novel taxane and
camptothecin represented a landmark in cancer research because of their significant
37
anti-solid tumour efficacy (Wall et al., 1966). Curcumin from Curcuma longa was
found to be cytotoxic in nature to a wide variety of tumour cell lines of different
tissue origin. It acts at various stages of tumour cell progression. Menon et al. (1999)
has proved the antimetastatic potential of curcumin in mice model. Iscador, an
extract from semiparasitic plant Viscum album inhibited the lung metastatic colony
formation induced by B16F-10 melanoma (Antony et al., 1997). Sulforaphane, an
isothiocyanate rich in broccoli showed anti metastatic activity in mice model
(Thejass and Kuttan, 2006). Punarnavine, an alkaloid present in the plant Boerhaavia
diffusa Linn has shown to inhibit pulmonary metastasis C57BL/6 mice (Manu and
Kuttan, 2009).
In the present study we have tested the Vernonia cinerea extract and its major
terpenoid compound, Vernolide-A for their immumomodulatory, antitumour,
antimetastatic, antiangiogenic and pro-apoptotic activity. We also screened some
naturally occurring terpenoids such as Perillic acid, Nomilin and Oleanolic acid for
their antiangiogenic and pro-apoptotic activity.
1.10.1. Vernonia cinerea Less
Vernonia cinerea L. (Asteraceae) has many therapeutic uses in different traditional
medicine of the world. Different parts of the plant are of different therapeutic values.
To mention a few, the plant is used for malarial fever, worms, pain, infections,
diuresis, cancer, abortion, and various gastro intestinal disorders (Hsu, 1967; John,
1984; Jain, 1984; Singh, 1989; Bhattarai, 1991; Bajpai, 1995; Graigner, 1996). Latha
et al. (1998) reported that the alcoholic extract of the flower was shown to posses
anti-inflammatory activity in adjuvant induced arthritis of rats. Other Vernonia
species that shared some of these therapeutic values include: Vernonia brachycalyx,
Vernonia brasiliana, Vernonia herbacea, Vernonia subligera and Vernonia coloralia
(Benoit, 1996; Alves, 1997; Oketch-Rabah, 1998). Phytochemical analysis of
Vernonia cinerea showed the presence of steroids, triterpenoids, sesquiterpenes,
flavanoids and tannins (Abeysekera, 1999; Hall, 1979; Jakupovic, 1986; Mishra,
1993).
38
1.11. CHEMOPREVENTION BY NATURALLY OCCURRING
TERPENOIDS
Terpenoids are minor but ubiquitous components of our diet, and are considered
relatively non-toxic to humans (Akihisa et al., 2003). These compounds, therefore,
have the potential of being used as cancer chemopreventive agents. Terpenoids, also
referred to as terpenes, are the largest group of natural compounds. They are plant
secondary metabolites along with alkaloids and flavonoids. Many terpenes have
biological activities and are used for the treatment of human diseases. Terpenoids are
formed from five-carbon isoprene units (C5H8) and also called isoprenoids. Based on
the number of the building blocks, terpenoids are commonly classified as
monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and sesterterpenes (C25)
(Wang et al., 2005). These terpenoids display a wide range of biological activities. In
this review we focus on the usefulness of selected terpenoids which have anti cancer
properties.
1.11.1. Vernolide-A
Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal
plants used in traditional medicine for the treatment of inflammatory diseases.
Vernolides are major sesquiterpenoids reported in Vernonia cinerea L., and are
considered as the active principle (Kuo et al., 2003; Chea et al., 2006). Various SLs
have been demonstrated to execute their anti-cancer capability via inhibition of
inflammatory responses, prevention of metastasis, angiogenesis and induction of
apoptosis. All SLs contain a common functional structure, an α-methylene-γ-lactone
group, and this important chemical characteristic means that the thiol-reactivity of
SLs is an underlying mechanism responsible for their bioactivities (Zhang et al.,
2005). Vernolide-A (C21H28O7) is a potent sesquiterpene lactone present in this plant.
Biological evaluation showed that Vernolide-A has potent cytotoxicity against
human KB, DLD-1, NCI-661, and Hela tumour cell lines (Kuo et al., 2003).
1.11.2. Nomilin
Citrus limonoids were demonstrated to possess potential biological activities in
reducing the risk of certain diseases. Nomilin is a triterpenoid with putative
anticancer properties. Immunomodulatory activity of the naturally occurring
triterpenoid, was already reported. Nomilin enhanced the antibody titre and the
39
number of plaque forming cells (PFC) in the spleen and also remarkably inhibited
delayed type hypersensitivity reaction (DTH) (Raphael et al., 2003). Cytotoxic
activity of nomilin against two human cancer cell lines, SH-SY5Y neuroblastoma
and Caco-2 colonic adenocarcinoma, and a noncancerous mammalian epithelial
Chinese hamster ovary (CHO) cells were already studied (Poulose et al., 2006).
Nomilin, which is the more active enzyme inducer, was found to inhibit
benzo[a]pyrene (BP)-induced neoplasia in the forestomach of ICR/Ha mice. The
number of mice with tumours was reduced from 100 to 72%, and the number of
tumours per mouse was significantly decreased as a result of nomilin treatment.
These findings suggest limonoids as a class of regularly consumed natural products
and effective chemopreventive agents (Lam et al., 1989).
1.11.3. Oleanolic acid
Oleanolic acid (OA) is a triterpenoid compound, widely found in natural plants (Liu,
1995), and has been shown to be an active ingredient in producing biological effects.
Oleanolic acid has been isolated from more than 120 plant species (Wang et al.,
1992). The anti-inflammatory effect of oleanolic acid was first reported in 1960s.
Gupta et al. (1969) reported the inhibitory effects of OA on carrageenan-induced rat
paw edema and formaldehyde- induced arthritis. The anti-inflammatory effects of
oleanolic acid have also been confirmed in later studies (Singh et al., 1992; Takagi et
al., 1980; Yue et al., 1989). It has been reported that OA produce a wide variety of
anti tumour activity, including decrease in the incidence and multiplicity of
azoxymethane-induced intestinal tumour. Treatment of rats with oleanolic acid (200
ppm) in diet for 3 weeks decreased the incidence and multiplicity of azoxymethane-
induced intestinal tumour (Yoshimi et al., 1992). The use of triterpenoid, OA has
been recommended for skin cancer therapy in Japan (Muto et al., 1990). The effect
of naturally occurring triterpenoid, oleanolic acid on immune system was studied
using Balb/c mice. Oleanolic acid enhanced the specific antibody titre and the
number of plaque forming cells (PFC) in the spleen and also inhibited delayed type
hypersensitivity reaction (DTH) (Raphael et al., 2003).
1.11.4. Perillic acid
Perillic acid (PA) is a major metabolite of perillyl alcohol (POH), a naturally
occurring monoterpene. The immunomodulatory activity of perillic acid was studied
40
in Balb/c mice. Administration of this monoterpene, increased the total antibody
production, antibody producing cells in spleen, bone marrow cellularity and alpha-
esterase positive cells significantly compared to the normal animals indicating its
potentiating effect on the immune system (Raphael et al., 2003). Perillic acid (PA) is
effective against a variety of rodent organ-specific tumour models. PA has a
potential for use as radiosensitizers in chemo-radiation therapy of head and neck
cancers and should be further studied (Samaila et al., 2004). Antimetastatic potential
of perillic acid were studied in C57BL/6 mice (Raphael et al., 2003).
Recommended