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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

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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

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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

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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

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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.

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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).

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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

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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

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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,

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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).

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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

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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

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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.

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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).