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Cancer is a group of diseases of higher multicellular organisms. It is characterized by

alterations in the expression of multiple genes, leading to dysregulation of the normal

cellular program for cell division and cell differentiation (Raymond, 2007). This

result is an imbalance of cell replication and cell death that favors growth of a tumor

cell population. The characteristics that delineate a malignant cancer from a benign

tumor are the abilities to invade locally, spread to regional lymphnodes and to

metastasize to distant organs in the body. Clinically, cancer appears to be many

different diseases with different phenotypic characteristics. As a cancerous growth

progresses, genetic drift in the cell population produces cell heterogeneity in such

characteristics as cell antigenicity, invasiveness, metastatic potential, rate of cell

proliferation, differentiation state and response to chemotherapeutic agents.

Hallmarks of malignant diseases

Malignant neoplasms or cancers have several distinguishing features that enable the

pathologist or experimental cancer biologist to characterize them as abnormal. The

most common types of human neoplasms derive from epithelium, that is, the cells

covering internal or external surfaces of the body. These cells have a supportive

stroma of blood vessels and connective tissue. Malignant neoplasms may resemble

normal tissues, at least in the early phases of their growth and development.

Neoplastic cells can develop in any tissue of the body that contains cells capable of

cell division. Though they may grow fast or slowly, their growth rate frequently

exceeds that of the surrounding normal tissue. This is not an invariant property,

however, because the rate of cell renewal in a number of normal tissues (e.g.,

gastrointestinal tract epithelium, bone marrow and hair follicles) is as rapid as that of

a rapidly growing tumor. The term neoplasm, meaning new growth, is often used inter

changeably with the term tumor sign for a cancerous growth. However, the tumors are

of two types: benign and malignant. The ability to distinguish between benign and

malignant tumors is crucial in determining the appropriate treatment and prognosis of

a patient who has a tumor. The following are features that differentiate a malignant

tumor from a benign tumor:

1. Malignant tumors invade and destroy adjacent normal tissue, while benign tumors

grow by expansion, are usually encapsulated and do not invade surrounding tissue.

Benign tumors may push a side normal tissue and may become life threatening if they

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press on nerves or blood vessels or if they secrete biologically active substances, such

as hormones, that alter normal homeostatic mechanisms.

2. Malignant tumors metastasize through lymphatic channels or blood vessels to

lymph nodes and other tissues in the body. Benign tumors remain localized and do not

metastasize.

3. Malignant tumor cells tend to be ‘anaplastic’ or less well differentiated than normal

cells of the tissue in which they arise. Benign tumors usually resemble normal tissue

more closely than malignant tumors do. Some malignant neoplastic cells at first

structurally and functionally resemble the normal tissue in which they arise. Later, as

the malignant neoplasm progresses, invades surrounding tissues and metastasizes, the

malignant cells may be are less resemblance to the normal cell of origin. The

development of a less well-differentiated malignant cell in a population of

differentiated normal cells is some times called dedifferentiation. This term

dedifferentiation is probably a misnomer for the process, because it implies that a

differentiated cell goes back wards in its developmental process after carcinogenic

insult. It is more likely that the anaplastic malignant cell type arises from the progeny

of a tissue ‘‘stem cell’’ (one that’s till has a capacity for renewal and is not yet fully

differentiated), which has been blocked or diverted in its pathway to form a fully

differentiated cell.

4. Malignant tumors usually do grow more rapidly than benign tumors. Once they

reach a clinically detectable stage, malignant tumors generally show evidence of

significant growth, with involvement of surrounding tissue, over weeks or months,

whereas, benign tumors often grow slowly over several years. Malignant neoplasms

continue to grow even in starvation of the host. They press on and invade surrounding

tissues, often interrupting vital functions and they metastasize to vital organs, for

example, brain, spine, and bone marrow, compromising their functions and they

invade blood vessels, causing bleeding. The most common effects on the patient are

cachexia (extreme body wasting), hemorrhage, and inection. About 50% of terminal

patients die from infection. Differential diagnosis of cancer from a benign tumor or a

non-neoplastic disease usually involves obtaining a tissue specimen by biopsy,

surgical excision, or exfoliative cytology. The latter is an examination of cells

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obtained from swabbings, washings or secretions of a tissue suspected to harbor

cancer: the ‘‘Pap test’’ involves such an examination.

Classification of human cancers

The suffix oma, applied by itself to a tissue type, usually indicates a benign tumor.

Some malignant neoplasms, however, may be designated by the oma suffix alone;

these include lymphoma, melanoma, and thymoma (Rubin, 1973). Rarely, the oma

suffix is used to describe a non-neoplastic condition such as granuloma which is often

not a true tumor, but a mass of granulation tissue resulting from chronic inflammation

or abscess. Malignant tumors are indicted by the terms carcinoma (epithelial in origin)

or sarcoma (mesenchymal in origin) proceeded by the histologic type and followed by

the tissue of origin. Examples of these include adenocarcinoma of the breast,

squamous cell carcinoma of the lung, basal cell carcinoma of skin, and

leiomyosarcoma of the uterus. Most human malignancies arise from epithelial tissue.

Those arising from stratified squamous epithelium are designated squamous cell

carcinomas, whereas, those emanating from glandular epithelium are termed

adenocarcinomas. When a malignant tumor no longer resembles the tissue of origin, it

may be called anaplastic or undifferentiated. If a tumor is metastatic from another

tissue, it is designated, for example, an adenocarcinoma of the colon metastatic to

liver. Some tumors arise from pluripotential primitive cell types and may contain

several tissue elements. These include mixed mesenchymal tumors of the uterus,

which contain carcinomatous and sarcomatous elements, and teratocarcinomas of the

ovary, which may contain bone, cartilage, muscle, and glandular epithelium.

Neoplasms of the hematopoietic system usually have no benign counter parts. Hence

the terms leukemia and lymphoma always refer to a malignant disease and have cell

type designations such as a cute or chronic myelogenous leukemia, Hodgkin’s or non-

Hodgkin’s lymphoma. Similarly, the term melanoma always refers to a malignant

neoplasm derived from melanocytes.

Growth characteristics of malignant cells

Most cancers (other than those for which there is a dominantly inherited cancer

susceptibility gene) are acquired molecular genetic disease in which a single (or a

few) clone (s) of cells accumulate cellular genetic changes that progress to the full

blown cancer phenotype. Cancer can be characterized as a disease of genetic

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instability, altered cellular behavior and altered cell extracellular matrix interactions

(Klausner, 2002). These alterations lead to dysregulated cell proliferation and

ultimately to invasion and metastasis. There are interactions between the genes

involved in these steps. For example, the genes associated with loss of control of cell

proliferation may also be involved in genetic instability (rapidly proliferating cells

have less time to repair DNA damage) and tumor vascularization that leads to

dysregulated proliferation of cells, which in turn eats up more oxygen, creates

hypoxia and additional angiogenesis. Similarly, genes involved in tumor cell invasion

may also be involved in loss of growth control (invasive cells have acquired the skills

to survive in ‘‘hostile’’ new environments) and evasion of apoptosis (less cell death

even in the face of a normal rate of cell proliferation produces more cells). The

molecular genetic alterations of cancer cells lead to cells that can generate their own

growth promoting signals, are less sensitive to cell cycle check point controls, evade

apoptosis and thus have almost limit less replication potential. As will be come

clearer, these signaling pathways are interlinked. As was not initially realized, cancer

cells have multiple proliferate pathways and can by pass an interdiction of one or

more of these. This redundancy makes design of effective signal transduction targeted

chemotherapeutic drugs that target a single pathway very difficult indeed. Cancer

cells can also subvert the environment in which they proliferate. Alterations in both

cell–cell and cell–extracellular matrix interactions also occur, leading to creation of a

cancer facilitating environment. For example, a common alteration in epithelial

carcinomas is alteration of E-cadherin expression. E-cadherin is a cell–cell adhesion

molecule found on all epithelial cells. Cancer cells exhibit remarkable plasticity.

Malignant cells have the ability to mimic some of the characteristics of other cell

types as they progress and became less well differentiated. For example, cancer cells

may assume some of the structure and function of vascular cells (Klausner, 2002). As

cancer cells metastasize, they may eventually take on a new phenotype such that the

tissues of origin may be come unclear so called cancers of unknown primary site. The

phenotypic properties found in transformed cells in culture are related to malignant

neoplasia in vivo is discussed below.

Immortality of transformed cells in culture

Most normal diploid mammalian cells have a limited life expectancy in culture. For

example, normal human fibroblast lines may live for 50 to 60 population doublings

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(the ‘‘Hayflick index’’), but then viability begins to decrease rapidly unless they

transform spontaneously or transformed by oncogenic agents. However, malignant

cells, once they become established in culture, will generally live for an indefinite

number of population doublings, provided the right nutrients and growth factors are

present. It is not clear what limits the life expectancy of normal diploid cells in

culture, but it may be related to the continual shortening of chromosomal telomeres

each time cells divide. Transformed cells are known to have elevated levels of

telomerase that maintain telomere length. Transformed cells that become established

in culture also frequently undergo karyotypic changes, usually marked by an increase

in chromosomes (polyploidy), with continual passage. This suggests that cells with

increased amounts of certain growth promoting genes are generated and or selected

during continual passage in culture. The more undifferentiated cells from cancers of

animals or patients also often have an a typical karyology, thus the same selection

process may be going on in vivo with progression over time of malignancy from a

lower to a higher grade.

Decreased requirement for growth factors

Other properties that distinguish transformed cells from their non-transformed counter

parts are decreased density-dependent inhibition of proliferation (Abercrombie and

Heaysman, 1954) and the requirement for growth factors for replication in culture.

Cells transformed by oncogenic viruses have lower serum growth requirements than

do normal cells (Dulbecco, 1970). For example, 3T3 fibroblasts transformed by SV40,

polyoma, murine sarcoma virus, or rous sarcoma virus are all able to grow in a culture

medium that lacks certain serum growth factors, whereas, uninfected cells are not

(Ruddon, 1995). Cancer cells may also produce their own growth factors that may be

secreted and activate proliferation in neighboring cells (paracrine effect) or, if the

same malignant cell type has both the receptor for a growth factor and the means to

produce the factor, self- stimulation of cell proliferation (autocrine effect) may occur.

One example of such an autocrine loop is the production of tumor necrosis factor-

alpha (TNF-α) and its receptor TNFR1 by diffuse large cell lymphoma (Graeber and

Eisenberg, 2001). Co-expression of TNFa and its receptor are negative prognostic

indicators of survival, suggesting that autocrine loops can be powerful stimuli for

tumor aggressiveness and thus potentially important diagnostic and therapeutic

targets.

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Loss of anchorage dependence

Most freshly isolated normal animal cells and cells from cultures of normal diploid

cells do not grow well when they are suspended in fluid or a semi solid agar gel. If

these cells make contact with a suitable surface, however, they attach, spread, and

proliferate. This type of growth is called anchorage-dependent growth. Many cell

lines derived from tumors and cells transformed by oncogenic agents are able to

proliferate in suspension cultures or in a semi solid medium (methyl cellulose or

agarose) without attachment to a surface. This is called anchorage-independent

growth. This property of transformed cells has been used to develop clones of

malignant cells (Sanders and Burford, 1964). This technique has been widely used to

compare the growth properties of normal and malignant cells. Another advantage that

has been derived from the ability of malignant cells to grow in soft agar (agarose) is

the ability to grow cancer cells derived from human tumors to test their sensitivity to

chemotherapeutic agents and to screen for potential new anti cancer drugs (Alley et

al., 1991).

Loss of cell cycle control and resistance to apoptosis

Normal cells respond to a variety of suboptimal growth conditions by entering a

quiescent phase in the cell division cycle, the G0 state. There appears to be a decision

point in the G1 phase of the cell cycle, at which time the cell must make a

commitment to continue into the S phase, the DNA synthesis step, or to stop in G1

and wait until conditions are more optimal for cell replication to occur. If this waiting

period is prolonged, the cells are said to be in a G0 phase. Once cells make a

commitment to divide, they must continue through S, G2, and M to return to G1. If

the cells are blocked in S, G2, or M for any length of time, they die. The events that

regulate the cell cycle, called cell cycle check points. This loss of cell cycle check

point control by cancer cells may contribute to their increased susceptibility to

anticancer drugs. Normal cells have mechanisms to protect themselves from exposure

to growth- limiting conditions or toxic agents by calling on these check point control

mechanisms. Cancer cells, by contrast, can continue through these check points into

cell cycle phases that make them more susceptible to the cytotoxic effects of drugs or

irradiation. For example, if normal cells accrue DNA damage due to ultraviolet (UV)

or X-irradiation, they arrest in G1 so that the damaged DNA can be repaired prior to

DNA replication. Another check point in the G2 phase allows repair of chromosome

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breaks before chromosomes are segregated at mitosis. Cancer cells, which exhibit

poor or absent check point controls, proceed to replicate the damaged DNA, thus

accounting for persisting and accumulating mutations.

Changes in cell membrane structure and function

The cell surface membrane (plasma membrane) plays an important role in the

‘‘social’’ behavior of cells, that is, communication with other cells, cell movement

and migration, adherence to other cells or structures, access to nutrients in the

microenvironment, and recognition by the body’s immune system. Alterations of the

plasma membrane in malignant cells may be inferred from a variety of properties that

characterize their growth and behavior, for example, the loss of density-dependent

inhibition of growth, decreased adhesiveness, loss of anchorage dependence, and

invasiveness through normal tissue barriers. In addition, a number of changes in the

biochemical characteristics of malignant cells surfaces have been observed. These

include appearance of new surface antigens, proteoglycans, glycolipids, and mucins,

and altered cell–cell and cell–extracellular matrix communication.

Modification of extracellular matrix components

The ECM plays a key role in regulating cellular proliferation and differentiation. In

the case of tumors, it is now clear that development of a blood supply and interaction

with them mesenchymal stroma on which tumor cells grow are involved in their

growth, invasive properties, and metastatic potential. This supporting stromal

structure is continuously remodeled by the interaction between the growing tumor and

host mesenchymal cells and vasculature. About 80% of the cells within a tumor are

stromal cells, including fibroblasts, non-tumor epithelial cells, mast cells, and

macrophages. The ECM components include collagen, proteoglycans, and

glycoproteins, such as fibronectin, laminin, and entactin. The ECM forms the milieu

in which tumor cells proliferate and provides a partial barrier to their growth.

Basement membranes are a specialized type of ECM. These membranes serve as a

support structure for cells, act as a ‘‘sieving’’ mechanism for transport of nutrients,

cellular metabolic products, and migratory cells (e.g. lymphocytes), and play a

regulatory role in cell proliferation and differentiation (Yurchenco and Schittny,

1990). Basement membranes also prevent the free passage of cells across them, but

there are mechanisms that permit the passage of inflammatory cells. It is also clear

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that basement membranes act as regulators of cell attachment, through cellular

receptors called integrins. There is also ‘‘cross talk’’ between epithelial cells and their

ECM to create a microenvironment for accurate signal transduction for growth factors

and other regulatory molecules. It has been shown, for example, that exogenous

reconstituted basement membranes stimulate specific differentiation of a variety of

cell types, including mammary cells, hepatocytes, endothelial cells, lung alveolar

cells, uterine epithelial cells, sertoli cells, and Schwann cells (Streuli and Bissell,

1990). The basement membrane barrier can be breeched by tumor cells that release a

variety of proteases, glycosidases, and collagenases that have the ability to degrade

various components of the matrix and thus allow tumor cells to invade through tissue

barriers and blood vessel and lymph channel walls. In addition, malignant cells them

selves have receptors for and or can produce certain components of the matrix; this

capability enables them to bind to the vascular endothelium and may be involved in

their ability to metastasize. Tumor cells may also release poly peptide factors that can

modulate the type of proteoglycans produced by host mesenchymal cells. The tumor

stromal cells, in turn, can release factors that favor tumor cell proliferation and

invasiveness. For example, activated fibroblasts in the tumor stroma release a number

of growth factors that stimulate cell proliferation, inhibit apoptosis, and alter cell

differentiation and that up-regulate proteases involved in degrading the ECM

(Bhowmick et al., 2004; Mueller and Fusenig, 2004). These factors include

hepatocyte growth factor (HGF), insulin like growth factors (IGF)-1and-2, EGF,

TGF-α, TGF-ß, inter leukin-6, fibroblast growth factors (FGF)-2 and -10, and matrix

metalloproteases-1 and 7 (Bhowmick et al., 2004). These multiple effects of the

tumor stroma on cancer growth and progression provide a number of potential targets

for anticancer therapy (Joyce, 2005).

Cell-extracellular matrix and cell – cell adhesion

Cells in tissues are attached to one another and to the ECM. Disruption of these

adhesion events leads to increased cell motility and potential invasiveness of cells

through the ECM. In addition, most cell types require attachment to the ECM for

normal growth, differentiation, and function. This attachment is responsible for what

is termed anchorage dependence. Normal cells that are detached from their binding to

the ECM under go apoptosis, whereas, tumor cells that are less dependent on this

attachment are free to proliferate, wander, and invade tissues. Cell adhesion to the

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ECM is mediated by cell surface receptors called integrins. Integrins are a family of

proteins consisting of αβ heterodimers that are integral membrane proteins with a

specific arginine, glycine and aspartic acid (RGD) amino acid sequence involved in

binding to the ECM (Ruoslahti and Pierschbacher, 1987). Integrins also link the

external ECM cytoskeleton to the intracellular actin cytoskeleton, and via this

connection a linkage to control of gene expression in the cell nucleus is established. In

this way, cell-ECM interactions can control gene read-out involved in cell

differentiation and function. Cell–ECM interactions occur via focal adhesions that

consist of clusters of ECM-bound integrins, and these in turn connect to actin fibrils

and the signal transduction machinery inside the cell. These signaling pathways

include the focal adhesion kinase (FAK) pathway that participates in the control of

anchorage dependence, and growth factor signaling pathways, such as the ras-raf-

mitogen-activated kinase, protein kinase C, and phosphatidylinositol 3-kinase

pathways (Giancotti and Ruoslahti, 1999). Thus, integrins cooperate with growth

factors to enhance mitogenic signaling. Alterations in integrin receptor expression

have been observed in chemically transformed human cells and in human colon and

breast cancer tissue (Ruddon, 1995). Cell-cell interactions are also important for the

normal regulation of cell proliferation and differentiation. These interactions are

mediated by a family of molecules called cell adhesion molecules (CAMs), which act

as both receptors (on one cell) and ligands (for another cell). The expression of CAMs

is programmed during development to provide positional and migratory information

for cells. A large family of CAMs has been identified. One group of these, called

cadherins, comprise a super family of Ca2þ dependent transmembrane glycoproteins

that play an essential role in the initiation and stabilization of cell-cell contacts.

Regulation of cadherin-mediated cell–cell adhesion is important in embryonic

development and maintenance of normal tissue differentiation (Stewart and Nelson,

1997; Uemura, 1998). The extracellular domain of various cadherins is responsible

for cell–cell homotypic binding (a given cadherin domain for a given cell type), and

the conserved cytoplasmic domains interact with cytoplasmic proteins called catenins.

Each cadherin molecule can bind to either b-catenin or g-catenin, which in turn bind

a-catenin. α-catenin links the cadherin complex to the actin cytoskeleton. Cell lines

that lack α-catenin lose normal cell-cell adhesiveness, and tumor cells with mutated or

down-regulated α-catenin have increased invasiveness (Vermeulen et al., 1995).

E-cadherin is the predominant type of cadherin expressed in epithelial tissue.

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Alterations of E-cadherin expression and function have been observed in human

cancers (Guilford, 1999). In addition, down-regulation of E-cadherin correlates within

increased invasiveness, metastasis, and poor prognosis in cancer patients. Suppression

of this invasive phenotype can be achieved by transfection of E-cadherin cDNA into

carcinoma cells, and contrarily, invasiveness of E-cadherin gene-transfected cells can

be restored by exposure of the cells to E-cadherin antibodies or an E-cadherin

antisense RNA (Guilford, 1999).

The cell surface receptor for E-cadherin is b-catenin. Early mutations in the human

colon cancer progression pathway affect the cellular distribution of ß-catenin. In

patients with colon cancer, the normal colonic epithelial cells adjacent to neoplastic

lesions had mostly cell surface membrane expression of ß-catenin, whereas,

cytoplasmic expression of b-catenin was observed in aberrant crypt foci (Hao et al.,

2001). Nuclear expression was observed in more advanced dysplasias and increased

as adenomas progressed to carcinomas. These latter changes are also observed in less

well-differentiated area soft tumors and are accompanied by loss of E-cadherin

expression at the invasive front of breast carcinomas, possibly due to

hypermethylation of the E-cadherin promoter (Brabletz et al., 2001).

Hyaluronic acid (HA)

Hyaluronan, a component of extracellular matrix (ECM) is a high-molar-mass linear

glycosaminoglycan (GAG) and simplest of all GAG’s. HA also found intracellularly

and on the surface of cells. HA can reach a size of 6 to 8 kDa. It is a ubiquitous

polymer with the repeating disaccharide structure of (β 1,3 N-acetyl-D-glucosamine -

β 1,4-D-glucuronic acid-)n. It has one carboxyl group per disaccharide repeating unit,

and is therefore a polyelectrolyte with a negative charge at neutral pH. It is near

perfect in chemical repeats, with no known deviations in its simple disaccharide

structure with the possible exception of occasional deacetylated glucosamine residues.

Hylauronic acid was first isolated from vitreous body of the eye (Meyer and Palmer,

1934). For decades, only few investigators were working on this polymer and the field

was not competitive. Now the situation has been changed in the last 10 years. Interest

in the hyaluronan has intensified in cell-biology, molecular biology, pathology,

immunology, after it was shown that cells have receptor that can specifically

recognizes its pure polysaccharide. Fig 1.1 shows the structure of hyaluronic acid.

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Fig.1

Structure of hyaluronic acid

(Source: http://www.madsci.org)

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The chemical structure of hyaluronan was determined in the 1950s in the laboratory

of Karl Meyer and other co-workers (Brimacombe and Webber, 1964).

HA biosynthesis

The discovery of three members of the HAS gene family (HAS-1, HAS-2, and HAS-

3) has enabled great strides in understanding the unique process of HA biosynthesis

and mode of chain elongation (Weigel et al., 1997; Itano and Kimata, 2002).

Structurally, all HAS proteins are composed of multiple membrane spanning regions

and large cytoplasmic loops (Fig.1.2). Unlike typical glycosyltransferases, the

cytoplasmic loop in HAS molecules possesses two active sites which participate in the

transfer of UDP-GlcNAc and UDP-GlcA substrates.

Characterization of the three HAS isoforms has revealed differences in enzymatic

properties, particularly in their ability to form HA matrices and determine product

size (Itano et al., 1999). The expression profiles of HAS genes are temporally and

spatially regulated during embryogenesis and pathogenesis (Sugiyama et al., 1998;

Kennedy et al., 2000; Recklies et al., 2001; Pienimaki et al., 2001), and divergence in

the transcriptional regulation of HAS genes during these processes can be explained

to some extent by upstream signaling pathways that are triggered by various growth

factors, cytokines, cellular stress, etc. The dynamic turnover of HA is therefore tightly

regulated by altering the expression profiles of HAS isoforms to have different

enzymatic properties (Weigel et al., 1997; Itano and Kimata, 1998, 2002). HAS has

very high rate of turnover where 5 g is replaced daily with half-life of about one day

and 2-5 min in blood system.

At the cellular level, a burst of HA synthesis occurs just prior to mitosis, enabling

some cells to become dissociated from neighboring cells and to lose the adhesion

from their surrounding ECM in preparation for division (Toole et al., 1972; Tomida et

al., 1974; Mian, 1986; Brecht et al., 1986). It is during this short period within the cell

cycle that normal cells most closely resemble transformed cells. The deposition of HA

preceding mitosis promotes detachment, and also confers motility directly upon cells

(Turley and Torrance, 1985; Turley et al., 1985), correlating possibly with the

movement of metastatic tumor cells.

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Fig.1.2

Scheme illustrating of HA structure (A), a predicted structure of

mammalian HAS (B), and a proposed secretion process of HA (C)

(Ref: Itano and Kimata, 2008)

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This large polymer have great deal of biological functions, as a glue it participates in

lubricating joints, space filling, anti angiogenic, immunosuppressive (McBride and

Bard, 1979; Feinberg and Beebe, 1983; Delmage et al., 1986;) and that impede

differentiation possible by suppressing cell-cell interactions or ligand access to cell

surface receptors. Hyaluronan chains can reach up to 2x104

(Laurent and Fraser,

1992) in size which are also involved in the ovulation, embryogenesis, protection of

epithelial layer integrity, wound repair and regeneration and their apparent size is

even greater when the solvent volume of surrounding water is considered. In general

it maintains the whole tissue integrity.

Hyaluronan has remarkable hydrodynamic characteristics, especially in terms of its

viscosity and its ability to retain water. It therefore has an important role in tissue

homeostasis and biomechanical integrity. Hyaluronan polymers are very large and can

displace a large volume of water. This property makes them excellent lubricators and

shock absorbers.

Hyaluronan also forms a multivalent template for interactions with proteoglycans and

other extracellular macromolecules that are important in the assembly of extracellular

and pericellular matrices (Toole, 2004). These properties of hyaluronan help to

regulate the porosity and malleability of these matrices which are important factors in

determining whether cells invade tissues during development, tissue remodelling and

cancer progression. This function of hyaluronan and of pericellular matrices

contributes to the ‘permissive’ or ‘landscaping’ role of the microenvironment in

which cancer cells proliferate and metastasizes (Hanahan and Weinberg, 2000).

Hyaluronan in cancer

HA deposition is up-regulated in most malignancies. Most of the malignant solid

tumors contain elevated levels of hyaluronan products (Knudson et al., 1989), which

are correlated with poor differentiation of cells (Auvinen et al., 1997). Enrichment of

hylauronan in tumors is due to the increase production of hylauronidase enzymes by

tumors cells themselves. Some malignant cells secrete or present membrane-bound

activities stimulating HA synthesis in adjacent fibroblasts (Knudson et al., 1984).

Stimulated HA synthesis by cancer cells or stromal fibroblasts may force gaps through

connective tissue, creating space for the invading cancer cells (Knudson et al., 1989).

It is shown that, elevated hyaluronan production by mouse mammary carcinoma

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(kimata et al., 1983) and melanoma cells (Zhang et al., 1995) correlates with

metastatic capacity. Besides, the interaction of several types of malignant tumor cells

with stromal cells up regulates stromal production of hyaluronan (Asplund et al.,

1993; Knudson et al., 1984; Knudson et al., 1989 and Toole et al., 1979).

High levels of stromal hyaluronan are also associated with malignancy in patients

who have non-small-cell lung adenocarcinoma (Pirinen et al., 2001) and prostate

cancer (Lipponen et al., 2001; Posey et al., 2003). Hyaluronan is predictive of

malignancy in some cancers (Toole et al., 2002) in breast and prostate carcinomas

hyaluronan is associated with tumor cells and stromal hyaluronan are linked with

cancer progression. Levels of parenchymal hyaluronan also correlate with malignancy

in patients with gastric and colorectal cancers (Toole, 2004). In addition, it has been

shown that hyaluronan levels are increased in the urine of patients with bladder

carcinomas (Lokeshwar et al., 1997), in the serum of patients with breast cancer

(Delpech, 1990) and in the saliva of patients with head and neck cancer (Franzmann

et al., 2003). However, hyaluronan levels do not correlate with progression in

melanomas (Karjalainen, 2000) or in some epidermal carcinomas (Karvinen et al.,

2003). Hyaluronan also constitutively regulates Erb2 phosphorylation and signaling

complex formation in colon and mammary carcinoma cells. Differential hyaluronan

expression in all human tumor progression explains the positive association of

increased stromal hyaluronan expression with invasive nature of tumors irrespective

of their origin (Boregowda et al., 2006).

Hyaluronan plays a critical role in dynamic structural changes within extracellular

matrices during development and tissue remodeling as well as maintenance of

mechanical properties and homeostasis of many tissues (Toole et al., 2002). Increased

synthesis of HA is associated with wound repair, tumor invasion and immune

recognition. Further, HA has been proposed to regulate cell locomotion and cyto-

differentiation that occur during these phenomena (Turley et al., 1985). HA is likely to

mediate these effects by sustained attachment to hyaluronan synthase across the plasma

membrane (Lee and Spicer, 2000; Toole, 2001; Turley et al., 2002) or by interacting

with its receptors, hyaluronan-binding proteins (HABPs) on cell surface such as CD44

(a trans-membrane receptor, Aruffo et al., 1990), TSG-6 (Lee et al., 1992), Receptor for

hyaluronic acid-mediated motility (RHAMM - a cell surface receptor, Hardwick et al.,

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1992), or with certain HA-receptors in the extracellular matrix, such as the aggregating

proteoglycan i.e., aggrecan, versican, brevican and neurocan.

Hyaluronan binding proteins

The hyaluronan-binding proteins (HABP’s) or hyaladherins, are a family of

macromolecules whose various members serve as structural components of

extracellular matrices or as receptors that bind hyaluronan to the surface of cells

(Toole, 1990). Many hyaladherins contain a common structural domain of ~100

amino acids in length, termed a Link module, that is involved in ligand binding (Day,

1999). However, a growing number of hyaladherins lack this domain and are

unrelated to each other at the primary sequence level. The widespread occurrence of

HA-binding proteins indicates that the recognition of HA is important to tissue

organization and the control of cellular behavior. Fig.1.3. shows the classification of

hyaladherins.

The hyaladherin family would include structural matrix HA-binding proteins as well

as cell-surface HA receptors that exhibit high affinity binding of HA. Most well

characterized hyaladherins have structurally similar hyaluronan binding domains with

sequence homologies of 30-40%. These domains are called link modules or

proteoglycan tandem repeats, form disulfide bonded loops and, in many hyaladherins,

two modules are arranged in tandem array. Two link modules form the hyaluronan-

binding region of link proteins and the aggregating proteoglycans, whereas, only a

single-link module is found in the hyaluronan-binding domains of CD44 (Sherman et

al., 1994) and TSG-6 (Lee et al., 1992). Some hyaladherins, such as RHAMM, do not

have link modules. However, mutation and sequence-swapping studies with RHAMM

showed a possible hyaluronan-binding motif that is present not only in RHAMM, but

also within or adjacent to the link modules of above explained hyaladherins.

The motif is B (X7) B, where B is arginine or lysine and X is any non-acidic amino acid.

Variations of this motif, e.g., B (X8) B, also bind to hyaluronan with significant affinity,

and clearly clustering of basic amino acids within and around the motif is the key

aspect that determines binding (Yang et al., 1994). Examples for the hyaluronan

binding proteins which contain the B(X7)B and related sequence are ICAM

(McCourt et al., 1994), hyaluronan synthases (Weigel et al., 1997), mammalian

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Fig. 1.3

Classification of hyaladherins

(Source: http://www.glycoforum.gr.jp/index.html)

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18

hyaluronidases (Zhu et al., 1994), Cdc37, a hyaluronan binding cell cycle regulatory

protein (Grammatikakis et al., 1995), P32, a hyaluronan-binding protein that

associates with splicing factors (Deb and Datta, 1996) and 1HABP4, an intracellular

hyaluronic acid binding protein (Huang et al., 2000). Fig. 1.3 shows the classification

of hyaladherins.

Hyaluronan binding proteins in cancer

Hyaluronan receptors have been widely implicated in tumorogenesis. CD44 and

RHAMM are established signal-transducing receptors that influence cell proliferation,

survival and motility, and are known to be relevant to cancer. Other cell-surface

hyaladherins such as lymphatic-vessel endothelial hyaluronan receptor 1 (LYVE1)

and TOLL4, might also have roles in cancer pathogenesis. Recent evidences suggest

that CD44 mediated events can enhance (Sy et al., 1991; Gunthert et al., 1991; Iida and

Bourguignon, 1997) or inhibit (Takahashi et al., 1995; Schmits et al., 1997) tumor

progression in different types of tumors.

CD44 (also known as homing cellular adhesion molecule, PGP-1, Hermes antigen,

and HUTCH-1) is a widely distributed type I transmembrane glycoprotein receptor

that binds primarily to the extracellular glycosaminoglycan, hyaluronan. CD44 is

encoded by a single gene but expressed as numerous isoforms as a result of alternative

splicing. The engagement of CD44 by hyaluronan results in intracellular signaling

that has been linked to such diverse effects as cellular adhesion, migration, and

invasion which are important in cancer progression, as well as hematopoietic

development and wound healing (Toole, 2002; Kosaki et al., 1999; Naor et al., 2002).

The standard form of CD44 (CD44s) is widely expressed and hyaluronan is

ubiquitous to most extracellular spaces, variant isoforms of CD44 have restricted

expression to specific conditions such as transformation, wound healing, and

lymphocyte activation (Naor et al., 2002).

Although hyaluronan is the main ligand for CD44, several other molecules interact

with this protein, many of which bind to carbohydrate side groups that are attached to

the ‘spliced-in’ regions. Among these other ligands, fibroblast growth factors,

osteopontin and matrix metalloproteinases (MMPs) are particularly important in terms

of relevance to cancer (Ponta et al., 2003). CD44 also mediates the cellular uptake

and degradation of hyaluronan, which in turn affects growth regulation and tissue

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Fig. 1.4

Genomic structure and multiple transcription products of the

CD44 gene (A) and Schematic drawing of the CD44 protein

The genomic structure is shown at the top (A) with the leader peptide (LP) and

transmembrane domain (TM) indicated. The variant exon nomenclature is notated

above exons 5a-14. Below (b-h) are some (but not all) observed transcripts from the

CD44 gene.

(Ref: Ponta et al., 1998)

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20

integrity (Kaya et al., 1997, Teder et al., 2002). Interactions of hyaluronan with CD44

lead to numerous cellular responses, including those that involve tyrosine kinases,

protein kinase C, focal adhesion kinase (FAK), phosphatidylinositol 3-kinase

(PI3K), mitogen-activated protein kinase, nuclear factor-κB and RAS, as well as

cytoskeletal components (Turley et al., 2002, Ponta et al., 2003, Bourguignon et al.,

2001, Thorne et al., 2004). Fig. 1.4 shows the Genomic structure and multiple

transcription products of the CD44 gene and Schematic drawing of the CD44 protein.

CD44 is a highly polymorphic protein due to the potential insertion of 10 variant

exons into the extracellular portion of the protein (CD44v). There are a total of nine

variant regions that can be coded for, v2–v10 (vl was isolated in rats and is not

present in human DNA). Variant CD44 (CD44v) can contain one or more variant

regions, such as CD44v6 or CD44v3–v7.

Many combinations of these variant exons are possible and individual cells can

repeatedly change the splicing of CD44 pre-mRNA, giving the potential for great

diversity. CD44v appears to have a much more restricted distribution and is expressed

on a variety of epithelial cells in a tissue specific pattern, suggesting that the process

of alternative splicing is normally tightly regulated. This also suggests that these

isoforms, with a specific exon sequence and restricted distribution, have different,

additional functions to CD44s (Sleeman et al., 1996). CD44v variants are also

involving in tumor progression and metastasis.

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HA oligosaccharides

Hyaluronidases, also called ‘spreading factor’, are a class of enzymes that

predominantly degrade hyaluronic acid (HA) to generate polymers of decreasing size.

Despite their simple size, HA oligosaccharides have extraordinary different biological

functions. The hyaluronidases fall into three classes (Meyer, 1971) based on analyses

of the reaction products:

1. Bacterial hyaluronidases (EC 4.2.99.1) are endo-β-acetyl-hexosaminidases

that function as eliminases yielding disaccharides. In marked contrast with

their eukaryotic counter parts, they are specific for HA.

2. Endo- β –glucuronidase types of hyaluronidase (EC 3.2.1.36) found in leeches,

crustaceans (Karlstam et al., 1991) and some parasites, generate tetra- and

hexasaccharide end products

3. The mammalian types of hyaluronidase (EC 3.2.1.35) are also endo- β-acetyl-

hexosaminidases, but function as hydrolases, with tetrasaccharides as the

predominant end products. They lack substrate specificity, able to digest

chondroitin sulfates (CS), though at a slower rate. In addition, they have

transglycosidase activity that generates complex cross-linked chains in vitro.

This ability has not been documented in vivo.

Six hyaluronidases like sequences are present in the mammalian genome, resulting

probably from two duplication events, resulting in three genes. All are

transcriptionally active with unique tissue distributions. In the human, three genes

(Hyal 1, Hyal 2, and Hyal 3) are found tightly clustered on chromosome 3p21.3,

coding for Hyal 1, Hyal 2, and Hyal 3. Another three genes (Hyal 4, PHyal 1, a

pseudogene), and SPAM1 (Sperm adhesion molecule1) are clustered similarly on

chromosome 7q31.3. They code respectively for Hyal 4, a pseudo gene transcribed

but not translated in the human, and PH-20, the sperm enzyme (Csoka et al., 1999;

2001). The enzymes Hyal 1 and Hyal 2 constitute the major hyaluronidases for HA

degradation in somatic tissues.

Hyal 1, an acid-active lysosomal enzyme, was the first somatic hyaluronidase to be

isolated and characterized (Afify et al., 1993, Frost et al., 1997). It is a 57 kDa single

polypeptide glycoprotein that also occurs in a processed 45 kDa form, the result of

INTRODUCTION

22

two endo protease reactions. The resulting two chains are bound by disulfide bonds.

This is not a zymogen active enzyme relationship, since the two isoforms have similar

specific activities. Why two forms should occur is unknown. Only the larger form is

present in the circulation, while both isoforms occur in urine (Csoka et al., 1997), in

tissue extracts, and in cultured cells. Why an acid-active hyaluronidase should occur

in plasma is not clear. Some species do not have detectable enzymatic activity in their

circulation (Fiszer et al., 1990), but an inactive 70 kDa precursor form of the enzyme

is present in such sera, detectable by Western blot. Hyal 1 is able to utilize HA of any

size as substrate, and generates predominantly tetrasaccharides. Fig. 1.5 shows the

putative scheme of HA catabolism.

Degradation begins when extracellular high-molecular-mass HA polymers of the

ECM are tethered to the cell surface through the combined action of CD44 (Culty et

al., 1992) and Hyal2 (Bourguignon et al., 2004). Receptors other than CD44 may be

involved, perhaps on a tissue- specific basis. In some cells, binding occurs with the

assistance of Na+–H+ exchanger 1 (NHE1) (Bourguignon et al., 2004). It has not

been documented whether or not most cells utilize this NHE1 mechanism. Hyal2 is a

glycosylphosphatidylinositol (GPI)-linked enzyme attached to the external surface of

the plasma membrane (Lepperdinger et al., 2001; Rai et al., 2001). The enzyme

makes the initial cleavage in high molecular mass HA, generating 20 kDa sized

products of approximately 100 saccharides (Lepperdinger et al., 1998). The HA

fragments are delivered to early endosomes, and to lysosomes where fragmentation

continues through the action of acid-active Hyal1, generating predominantly

tetrasaccharides (Fig.1.5). Additionally, despite all these details, it remains unclear at

what point the fragmentation of HA switches from an extracellular or cell surface

process to an endosomal or lysosomal process. This HA fragments shows

extraordinary wide ranging biological functions, which may also depend on the

cellular up take were the specific receptors exists for the variable size HA chains.

HA oligosaccharides in normal and pathological process:

Angiogenesis, and wound healing

HA of lower molecular size accumulates in wound healing. The mononuclear cells,

including monocytes and lymphocytes, now appear at the wound site. Expression

of inflammatory cytokines is induced by HA fragments in the 1000–1250 saccharide

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Fig.1.5

A putative scheme for HA catabolism

(Ref: Stern, 2004)

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24

range (200–250kDa). The function of this size of fragments was first examined in in

vitro studies (Noble et al., 1993, 1996, 2002; Hodge-Dufour et al., 1997; Horton et

al., 1999; McKee et al., 1993, 1997). Similar results have been shown with renal

tubular epithelial cells (Beck-Schimmer et al., 1998), cancer cells (Fitzgerald et al.,

2000) and eosinophils (Ohkawara et al., 2000). The inflammatory stage of wound

healing is followed and overlaps partially with an angiogenic response. The first

report of HA oligomers involvement in angiogenesis appeared in 1985, in which HA

fragments limited to the 6–20 size range were shown to be angiogenic (West et al.,

1985, West and Kumar, 1989; Rooney et al., 1995; Horton et al., 1998, 2000). These

HA fragments are not only mitogenic for endothelial cells (Slevin et al., 1998, 2002),

but also enhance endothelial cell migration and induce multiple signaling pathways

(Sattar et al., 1994; Slevin et al., 2002). Such HA fragments induce tyrosine kinase

cascades (Lokeshwar and Selzer, 2000). Subsequent to the angiogenic response is the

proliferation of fibroblasts, involved in the final stage of the repair process. HA

fragments similar to those involved in the angiogenic response, in the 6–20 range of

saccharides, stimulate fibroblast proliferation and synthesis of collagen (Rooney et al.,

1993). HA fragments of a smaller size, in the 4–6 saccharide range, stimulate cytokine

production by dendritic cells, the antigen-presenting cells of the immune system

(Termeer et al., 2000, 2002).

HA oligosaccharides in cancer

HA fragments are having various biological functions. A partial list of HA fragments

and their biological functions correlated with molecular size is given in Table 1.1.

High molecular mass HA is found in most normal biological processes, much lower

weight material is readily detected in cancers (Kumar et al., 1989; Lokeshwar et al.,

1997), where it facilitates tumor cell motility and invasion. HA oligosaccharides of a

certain size range induce proteolytic cleavage of CD44 on the surface of cancer cells,

and promote tumor cell migration in a CD44 and dose-dependent manner (Sugahara et

al., 2003, 2004), reinforcing the concept that HA fragments facilitate cancer

progression. Tumor cells can also secrete constitutively abundant amounts of Hyal

activity, generating HA fragments in the 10–40 saccharide range, enhancing cleavage

of their own CD44 and their own motility. Such an autocrine mechanism can promote

malignant progression in the absence of external stimulation (Sugahara et al., 2005).

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Table 1.1

A partial list of HA fragments and their assigned biological functions correlated

with molecular size

Size (saccharides) Function Reference: High molecular- mass

HA >1000-5000

HA fragments -1000

10-40

8-32

~15

12

10

4-6

4

Suppression of angiogenesis

Immune suppression

Inhibition of phagocytosis

Suppression of HA synthesis

Induction of inflammatory chemokines

Stimulation ofPAI-1

Stimulation of urokinase

Induction of CD44 cleavage

Promotion of tumor cell migration

Stimulation of angiogenesis

Stimulation of tumor neovascularization

Suppression of smooth muscle cell proliferation

Endothelial cell differentiation

Up-regulation of PTEN in tumor cells

Displacement of matrix HA on oocyte surface

Displacement of proteoglycans from cell surface

Suppression of HA cable formation Induction of

NO and MMPs in chondrocytes

Induction of HAS2 in chondrocytes Induction of

cytokine synthesis in dendritic cells

Transcription of MMPs

Up- regulation of Hsp 72 expression

Suppression of apoptosis

Induction of chemotaxis

Up-regulation of heat shock factor-1

Up-regulation of Fas expression

Suppression of proteoglycan sulfation

Feinberg and Beebe, 1983

McBride and Bard, 1979,

Delmage et al., 1986

Forrester and Balazs, 1980

Lueke and Prehm, 1999

Noble et al., 1993

Horton et al.,2000

Horton et al., 2000

Sugahara et al., 2003

Sugahara et al., 2003

West et al.,1985; Sattar et al., 1994;

Slevin et al., 1998, 2002

Rooney et al., 1995

Evanko et al., 1999

Takahashi et al., 2005

Ghatak et al., 2002

Camaioni et al., 1993

Solursh et al., 1980

De la Motte et al., 2003

Knudson and Knudson, 2004

Termeer et al., 2000, 2002

Taylor et al., 2004

Fieber et al., 2004

Xu et al., 2002

Xu et al., 2002

R. Savani, personal communication

Xu et al., 2002

Fujii et al., 2001

Solursh et al., 1980

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The size range of HA fragments that induce cleavage of CD44 in cancer cells have

been care fully investigated. Added exogenously to cultured pancreatic cancer cells,

6–14 saccharides induce maximal cleavage in a dose dependent manner. The cleaved

CD44 is released into the circulation (Stern et al., 2006). This may be part of a

strategy for the cancer cell to become independent of CD44-related controls, by

providing a circulating form of the molecule that competes with cell surface CD44.

Increased CD44 cleavage has been documented in gliomas, breast, colon and ovarian

cancers, and in non-small cell carcinomas of the lung (Okamoto et al., 1999). Highly

invasive bladder cancers produce HA fragments in the 30–50 saccharide range, the

sizes that are angiogenic for endothelial cells though larger in size than those reported

earlier (Lokeshwar et al., 1997). This is perhaps the mechanism for their invasiveness

and serves as an example of how malignancies can commandeer normal physiological

functions for their own purposes. The 35 saccharide size fragments of HA activate

tumor cell integrins, enhancing cell binding to intercellular adhesion molecule-1

(ICAM-1) (Fujisaki et al., 1999). By contrast, the HA fragments ranging from 6 to 24

saccharides inhibit B16F10 melanoma cell proliferation in vitro, as well as the

formation of tumors from subcutaneously injected cells in vivo (Zeng et al., 1998).

Very small HA oligosaccharides also have unique biological activities. HA

oligosaccharides in the 6–7 saccharide range inhibit expression of matrix

metalloproteinases (MMPs) in cancer cells, specifically MMP-9 and MMP-13

(Fieber et al., 2004).

Thus, the oligosaccharides of HA can either promote or inhibit tumor progression.

The confusion and inconsistencies that abound can be attributed to the adage that

different tumors do different things and the same tumor can do different things at

different times. Instability of the tumor genome and the constant Darwinian selection

process of tumor metastases underscore the resilience and ingenuity of malignant cells

in their ability to survive and thrive. This applies apparently also to the widely

differing ability to generate different sizes of HA fragments.

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HA oligosaccharides in signal transduction

Several signal transduction pathways are initiated by various sizes of HA fragments

binding to cell surface HA receptors such as CD44 and RHAMM. Table1.2 provides

a sample of only some of the transduction pathways that have been documented.

Angiogenic oligosaccharides of HA induce tyrosine kinases in endothelial cells and

activate several cytoplasmic signaling transduction pathways such as Raf-1 kinase,

MAP kinase and extracellular signaling kinases such as ERK-1, all of which result in

proliferation (Slevin et al., 1998). HA-CD44 interactions stimulate ERK signaling and

transcriptional activation in ovarian cancers (Bourguignon et al., 2005) as well as

promoting drug resistance in head and neck cancers through phospholipase C-

mediated Ca2+ signaling (Wang and Bourguignon, 2006). There is, as well,

phosphorylation of the CD44 receptor, increased levels of protein kinase C and

translocation of phospholipases within the plasma membranes (Slevin et al., 2002).

Oligosaccharides of HA in the 6–20 range regulate Erb2 phosphorylation and

signaling in cancer cells (Ghatak et al., 2005). The ability of small oligosaccharides

such as hexamers to inhibit tumor growth can be attributed to PTEN, a phosphatase

that degrades PIP3, the product of PI 3 kinase action, inhibiting growth by inducing

pro-apoptotic mediators (Ghatak et al., 2002). Hyaluronan oligosaccharides in the 4–6

saccharide range also activate an NF-kB/I-kB alpha auto-regulatory loop (Noble et

al., 1996), inducing transcription of metalloproteases MMP-9 and -13 (Horton et al.,

1999; Fieber et al., 2004).

The induction of nitric-oxide synthase by such saccharides also occurs through a

nuclear NF-kB-dependent mechanism (McKee et al., 1997).Such low-molecular-size

HA fragments are also malignant cells by endogenous or exogenous treatment with

HA-oligo induces apoptosis under anchorage-independent conditions and reduces

tumor growth. The hypothesis that HA-oligo acting as an antagonist for HA- cell

surface interaction would provide a direct relationship and to detect important

regulators of microglia at the site of ischemic brain damage (Wang et al., 2004),

where c-Jun N terminal kinase and p38 mitogen-activated protein kinase induce nitric

oxide synthase expression.

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Table-1.2

A sample of only some transduction pathways

that has been documented

Molecular size Signaling Molecules References

~ 4

4

6

~12

6-20

~34

Not

determined

1L-12, TNFa

Up-regulation of Fas expression

Erk, JNK, p38 stimulation

Activation of NF-KB in chondrocytes

Up-regulation of PTEN in tumor cells

Inhibition of anchorage-independent

growth through suppression of PI 3

kinase

FAK, PI 3 kinase

Activation of NF-KB

Termeer et al., 2002

Fujii et al., 2001

M. Tammi and R. Tammi,

personal communication

Knudson and Knudson,

2004

Ghatak et al., 2002

Ghatak et al., 2002

Fujita et al., 2002

Fitzgerald et al., 2000

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Size-specific binding of HA fragments to hyaladherins

HA fragments can bind to HA-binding proteins or hyaladherins (Toole, 1990;

Knudson and Knudson, 1993). Such binding has an array of functions, from

intracellular effects, such as regulators of the cell cycle (Grammatikakis et al., 1995)

or as splicing factors (Deb and Datta, 1996). Extracellular effects are provided by

binding to cell surface receptors such as RHAMM and CD44, or to extracellular

proteoglycans such as aggrecan and versican. Association with specific HA binding

proteins provides the structural integrity of many tissues by way of their extracellular

matrices, including cartilage, brain, and the walls of blood vessels (Day and

Prestwich, 2002; Seyfried et al., 2005).

Variations occur in the minimum size of HA oligosaccharides that bind to HA-

binding proteins. The HA chain takes on various secondary and tertiary structures that

are in part dependent on polymer size (Scott et al., 1984; Scott and Heatley, 2002).

The earliest among these studies (Hardingham and Muir, 1973; Hascall and

Heinegard, 1974) demonstrated that a 10-mer is the minimum size HA oligomer able

to bind strongly to the proteoglycan, aggrecan. The avidity of binding to CD44

increases with oligomer size up to 38 sugars (Lesley et al., 2000). Some of this

relationship between HA-oligomer size and HABP are summarized in Table 1.3.

HA-oligosaccharides have diverse and complex biological functions both in vivo and

In vitro (Slevin, 2002; Noble, 1996). Cellular functions of HA-oligosaccharides of

defined length can be used to characterize multiple proteins-HA interactions.

Octasaccharides can bind optimally to the link module of TSG-6 and HA-binding

domain of CD44. This binding may be different from HA-polymer binding to

aggrecan, link protein, versican by virtue of incomplete competition by small

oligosaccharides (Hascall and Heinegaard, 1974). More information is necessary to

understand how various sizes of HA-oligo and it receptor can influence the vital

function of cellular processes. HA-oligosaccharides produced due to endogenous

enzymes in freshly excised human tumor and its associated benign tissues are vitally

important to understand its effect in regulating tissue integrity. Mainly because of

perturbation of HA- cell interaction in multiple hyaladherins and other cell surface

proteins and will become a valuable tools in evaluating HA-oligosaccharides

involvement in cancer metastasis.

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Table1.3

Size specific binding of proteins to HA oligosaccharides

Molecular size

(saccharides)

Proteins References

6

8

8-10

10

50

HABP1

CD44 and TSG-6 link module

Chondrocyte CD44 Smooth muscle

cell CD44

Heavy chain of inter-a-trypsin inhibitor

TSG-6

SHAP

Aggrecan

Versican

Link protein

Keratinocyte CD44 (CD44E)

Link protein plus aggrecan

Deb et al., 2002;

Kohda et al., 1996; Knudson, 1993

de la Motte et al., 2003

Mukhopadhyay et al., 2004

Kahmann et al., 2000

Yoneda et al., 1990

Hascall and Heinegard, 1974

Seyfried et al., 2005

Seyfried et al., 2005

Tammi et al., 1998

Kimura et al., 1979

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So a close association exists between glycosaminoglycan, extracellular

polysaccharide hyaluronan and malignant tumor progression. Hyaluronan and its

binding proteins (receptors) are usually higher in metastatic tumors (i.e. gastric

cancer, colorectal cancer, breast cancer, glioma, lung cancer, ovarian cancer etc.) than

in corresponding benign or normal tissues (Toole 2002; Boregowda et al., 2006).

Increase of hyaluronan in extracellular matrix provides a ground for metastatic

capacity of tumor cells, stimulate cell interaction with HA-binding proteins and

forming a barrier for cancer cells against host immuno competent cells.

In the present study, an investigation was carried out to identify HA oligosaccharides

and its binding proteins and also studied their distribution by Immunohistochemistry

in human benign and malignant tumor tissues. Since, HA hexasaccharide having

several biological functions, an attempt has been made to characterize its interacting

receptors. This would provide evidence in understanding the involvement of HA

oligosaccharides binding proteins in progressive cancer tissue.

Objectives of the investigation:

Preparation and characterization of HA oligosaccharides present in benign and

malignant tumors

Detection and Purification of size specific HA oligo binding proteins from

normal and tumor tissues.

Characterization of size specific HA oligo binding proteins by determining

their homology with known HABPs.

Histochemical localization of size specific HA oligosaccharides in tumor

tissue sections.