glycodendrimer mediation by

GLYCODENDRIMER MEDIATION

OF GALECTIN-3 INDUCED CANCER CELL AGGREGATION

by

Julie Jeannine Sprenger

A thesis submitted in partial fulfillment

of the requirements for the degree

of

Master of Science

in

Biochemistry

MONTANA STATE UNIVERSITY

Bozeman, Montana

November 2010

©COPYRIGHT

by


2010

All Rights Reserved

ii

APPROVAL

of a thesis submitted by


This thesis has been read by each member of the thesis committee and has been

found to be satisfactory regarding content, English usage, format, citation, bibliographic

style, and consistency and is ready for submission to the Division of Graduate Education.

Dr. Mary J. Cloninger

Approved for the Department of Chemistry and Biochemistry

Dr. David Singel

Approved for the Division of Graduate Education

Dr. Carl A. Fox

iii

STATEMENT OF PERMISSION TO USE

In presenting this thesis in partial fulfillment of the requirements for a master‟s

degree at Montana State University, I agree that the Library shall make it available to

borrowers under rules of the Library.

If I have indicated my intention to copyright this thesis by including a copyright

notice page, copying is allowable only for scholarly purposes, consistent with “fair use”

as prescribed in the U.S. Copyright Law. Requests for permission for extended quotation

from or reproduction of this thesis in whole or in parts may be granted only by the

copyright holder.


November 2010

iv

ACKNOWLEDGEMENTS

Ultimate thanks goes to the Lord who has given me breath, life, an inquiring mind

and indispensable, insurmountable grace (Ephesians 2:8 ). Thank you to my parents for

the foundation you have laid, your prayers, your encouragement to venture out and

unconditional support. Thank you Mary for your selfless counsel, support, willingness to

go the extra mile to get me to MSU and get me through, and for your constant

encouragement. Your love of science has rubbed off on me and my desire is to show that

same passion to my students. An eternal thanks to my faithful “sisters” Jeanne and

Shannon. Thank you for holding me accountable, helping solve the world‟s problems and

being my „balcony people‟.

To all of the Cloninger lab members that I have had the privilege to work with,

you‟re lovely! I wish you all well in your future endeavors and continued success. Thank

you!

v

TABLE OF CONTENTS

1. AN INTRODUCTION TO GALECTIN-3 ................................................................... 1

Galectin/Carbohydrate Interaction ............................................................................... 1

Galectin-3 Structure and Biological Functions ............................................................. 2

Galectin-3 Ligands ...................................................................................................... 4

2. THE ROLE OF EXTRACELLULAR GALECTIN-3 IN CANCER ............................. 6

Galectin-3 Expression in Cancer Cells ......................................................................... 6

Galectin-3 in Metastasis ............................................................................................... 8

Primary Tumor Formation: Galectin-3 in Homotypic Cellular Aggregation.......... 9

Galectin-3 in Angiogenesis ................................................................................ 10

Localized Invasion, Intravasation and Extravasation: Galectin-3 in

Heterotypic Cellular Aggregation ....................................................................... 11

Formation of Micro Metastasis and Colonization: Galectin-3 in

Embolism and Secondary Tumor Formation ...................................................... 12

Summary of Galectin-3 in Cancer .............................................................................. 13

3. CARBOHYDRATE FUNCTIONALIZED SYSTEMS TO STUDY GALECTIN-3 ... 14

Introduction to Carbohydrate Functionalized Systems ................................................ 14

Carbohydrate-Functionalized Systems to Study Galectin-3/Carbohydrate

Interactions ................................................................................................................ 14

The Dendrimer Framework ........................................................................................ 19

Summary of Carbohydrate Functionalized Systems to Study Galectin-3 .................... 23

4. HOMOTYPIC AGGREGATION STUDIES ............................................................. 24

Background ............................................................................................................... 24

Goals ......................................................................................................................... 24

Project Description .................................................................................................... 25

Results ....................................................................................................................... 27

Analysis ..................................................................................................................... 40

5. SUMMARY, CONCLUSIONS AND FUTURE WORK ........................................... 44

6. MATERIALS AND METHODS ............................................................................... 46

Galectin-3 Purification ............................................................................................... 46

Carbohydrate Functionalized Dendrimers .................................................................. 47

Cell Cultures .............................................................................................................. 47

vi

TABLE OF CONTENTS – CONTINUED

Homotypic Cellular Aggregation Assay .................................................................... 47

REFERENCES CITED ................................................................................................. 49

vii

LIST OF TABLES

Table Page

3.1. Theoretical calculated properties of amine surface functional PAMAM

dendrimers by generation ............................................................................ 21

4.1 Concentrations of inhibitory compounds ...................................................... 41

viii

LIST OF FIGURES

Figure Page

1.1 Cell-surface glycoproteins interact with carbohydrate binding domains .......... 1

1.2. (a) The β-sandwich CRD of galectin-3

(b) Sub-binding sites in the S strand, A, B, C, D, and E, the core

binding sites bound with lactose .............................................................. 2

1.3 Galectin types................................................................................................. 3

1.4 A schematic representation of the cross linking of cells by galectin-3

interaction with cell surface carbohydrates and N-terminal self-association ... 4

1.5 Galectin ligands.............................................................................................. 5

3.1 Tumor growth reduction in mice treated with modified citrus pectin ............ 15

3.2 The effect of oligosaccaride derivates on galectin-3 induced homotypic

aggregation of A375 cells ............................................................................. 16

3.3 Structures of oligosaccharide derived galectin inhibitors .............................. 17

3.4 Tri-valent rigidified lactoside clusters tested for lectin inhibition .................. 18

3.5 The pseudopolyrotaxane. .............................................................................. 19

3.6 (a) A schematic representation of a dendrimer interacting with the active site

of galectin-3 and inhibiting cellular aggregation. (b) A schematic

representation of a dendrimer interacting with multiple galectin-3 proteins and

promoting aggregation ................................................................................. 20

3.7 (a) PAMAM dendrimer scaffold generations 0 (blue), 1 (red) and 2 (purple).

(b) Three dimensional representations of the “tree like” branching PAMAM

dendrimer .............................................................................................. 21

3.8 Functionalized dendrimers............................................................................ 22

4.1 Homotypic aggregation assay procedure. ...................................................... 26

4.2. Control homotypic aggregation assay cell pictures ...................................... 27

ix

LIST OF FIGURES - CONTINUED

Figure Page

4.3. The effects of increasing concentrations of lactose on galectin-3 induced

homotypic HT-1080 aggregation.. ............................................................... 29

4.4 Pictures of HT-1080 cellular aggregation with galectin-3 and lactose.. ......... 29

4.5 The effects of increasing concentrations functionalized dendrimer 1 on

galectin-3 induced homotypic HT-1080 aggregation.. ................................. 30

4.6 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized

dendrimer 1.. ................................................................................................ 30




dendrimer 1.. ................................................................................................ 31




dendrimer 1.. ................................................................................................ 32




dendrimer 1.. ................................................................................................ 33




dendrimer 2.. ................................................................................................ 34



x

LIST OF FIGURES - CONTINUED

Figure Page


dendrimer 2.. ............................................................................................... 35




dendrimer 2.. ................................................................................................ 36




dendrimer 4.. ................................................................................................ 37




dendrimer 5.. ................................................................................................ 38




dendrimer 8.. ................................................................................................ 39

4.25 (a) A schematic representation of the smaller generations of functionalized

dendrimers inhibiting cellular aggregation. (b) A schematic representation of

the larger generations of functionalized-dendrimers enhancing and varying

cellular aggregation. ..................................................................................... 43

xi

ABSTRACT

Galectin-3 is a cell surface protein that plays an important role in tumor

aggregation, tumor progression and metastasis via its interaction with carbohydrates

in the biological system. A synthetic, carbohydrate-functionalized, multivalent

framework is ideal to study biological protein/carbohydrate interactions. In this

research, dendrimers are used as a platform for the display of carbohydrates to study

multivalent galectin-3/carbohydrate interactions as they pertain to tumor aggregation.

The hypothesis is that the addition of carbohydrate functionalized dendrimers will

mimic natural galectin-3 glycoconjugate ligands, affecting the aggregating behavior

of neoplastic cells. A galectin-3 inhibitor may prove to be an effective cancer

therapeutic agent. Results from homotypic aggregation assays show a change in

aggregation with the addition of particular carbohydrate-functionalized dendrimers.

Two of the glycodendrimers significantly inhibit tumor cell aggregation.

1

CHAPTER 1

AN INTRODUCTION TO GALECTIN-3

Galectin/Carbohydrate Interaction

In many biological processes, cellular recognition and adhesion are mediated by

protein-carbohydrate interactions (Figure 1.1). Lectins, which are found in most

organisms, are a family of proteins that specifically recognize and reversibly bind mono-

and oligosaccharides. Lectins are capable of inducing cellular agglutination by cross-

linking cells through two or more carbohydrate binding sites. 1

Figure 1.1. Cell-surface glycoproteins interact with carbohydrate-binding domains.

Galectins are a class of lectins that contain at least one carbohydrate recognition

domain (CRD) that specifically binds β-galactosides.2 The CRD, shown in Figure 1.2 (a),

contains 130 amino acids arranged in a β-sheet sandwich. The domain binds

carbohydrates at the groove on the concave side of the β-sandwich, labeled S that is

formed by six strands. Figure 1.2 (b) is a close-up of the core binding site, labeled C-D,

showing the specific amino acid interactions with lactose. Six of the total seven

interacting amino acids interact with the galactose. The majority of these galactose

2

interactions are hydrogen bonds that originate from sub-site C and are conserved through

the galectin family.3 The amino acid interaction via sub-site D with glucose in the lactose

molecule is a partially conserved interaction, therefore the binding role can be fulfilled by

different saccharides. Sub-sites A, B, and E are sources of variation among galectins,

causing each galectin to prefer binding to different saccharides.4

(a) (b)

Figure 1.2. (a) The β-sandwich CRD of galectin-3. S - β sheets (thicker lines) and

F- β sheets. (b) Sub-binding sites in the S strand, A, B, C, D, and

E, the core binding sites bound with lactose (Galβ1- 4Glc).4

Galectin-3 Structure and Biological Functions

There are fifteen identified and isolated galectins. Galectins-1, -2, -5, -10, -11, -

13, -14, and -15 contain only one CRD, biologically acting as monomers or homodimers.

Galectin-3 is a chimera type galectin; it contains only one CRD but is unique among the

galectins because it contains a biologically active “non-lectin” N-terminal domain that is

responsible for the oligomerization of the protein giving galectin-3 multivalent

properties.2

Galectins- 4, -6, -8, -9, and -12, are tandem-repeat type galectins that contain

3

two CRD domains connected by a short linker peptide. Figure 1.3 depicts the physical

differences between the fifteen galectins.4

(a) (b) (c)

Figure 1.3. Galectin types. The CRD domain is in black. (a) Monomeric and

homodimeric galectins -1, -2, -5, -10, -11, -13, -14 and -15. (b) Chimeric galectin-3.

(c) Tandem-repeat type galectins -4, -6, -8, -9 and- 12.3

Galectin-3 is unique among the galectins with its non-lectin N-terminal domain.

Galectins typically induce cross-linking of cells and cell surface ligands through the

interactions of more than one carbohydrate binding domain. Galectin-3 self associates

through its glycine, proline and tyrosine rich, collagen like N-terminal tail, cross-linking

oligosaccharides on the cell surface inducing aggregation (Figure 1.4).5, 6

Fragments of

the purified N-terminal domain have been seen to associate7. Also, the carbohydrate

recognition domain alone, without the N-terminal domain, binds lactose but has no

agglutination activity.8 This oligomerization has been visualized with fluorescence

resonance energy transfer.6

4

Figure 1.4. A schematic representation of the cross linking of cells by galectin-3

interaction with cell surface carbohydrates and N-terminal self- association.

The ability of galectin-3 to readily interact with carbohydrates and to oligomerize

is the key driving force for most extracellular galectin-3 biological activities. Research

shows that extracellular galectin-3 mediates cell surface signal transduction, organ

development, neuronal functions, autoimmune disorders, endocytosis, cell to cell and

cell to extracellular matrix adhesion, angiogenesis, and tumor progression through ligand

crosslinking9. The focus of the research that is reported here is cell to cell adhesion

induced by galectin-3 as it relates to tumor progression.

Galectin-3 Ligands

The CRD domain of galectins binds galactose based carbohydrates, many of

which are aberrantly expressed on tumor cells. The ligands for galectins, which are

shown in Figure 1.5, include but are not limited to galactose, N-acetylgalactosamine,

lactose, N-acetyllactosamine, Tn antigen and Thomsen-Friedenreich antigen (Tf antigen).

Tf antigen, present on the cell surface of tumor cells, is considered a natural ligand for

galectin-3.10

5

Figure 1.5. Galectin ligands.

There are several factors that contribute to galectin-3 binding. The binding of

galectin-3 to simple β- galactosides is weak. The oligomeric, multivalent, state of

galectin-3 significantly increases its binding to ligands. Binding is also enhanced where

an increasing number of N-acetyllactosamine units are present.11

Physiologically,

galectin-3 interacts with high affinity to glycoconjugate ligands expressed on cell

surfaces or in the extracellular matrix such as basement membrane proteins including

laminin, integrins (membrane proteins), lysosome-associated membrane proteins,

vitronectin and fibronectin, and cancer-associated MUC1 via the Thomsen Friedenreich

(TF) antigen (Galβ1,3GalNAc-α Thr/Ser).11,

12

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6

CHAPTER 2

THE ROLE OF EXTRACELLULAR GALECTIN-3 IN CANCER

Galectin-3 Expression in Cancer Cells

Substantial evidence suggests that the level of galectin-3 expressed in a cell

strongly correlates with the neoplastic progression of several cancer types. Thyroid,

colorectal, gastric and human head and neck tumors all show increased galectin-3

expression as the cancer progresses.13

Galectin-3, which is not typically expressed in thyroid tissue, is over-expressed in

cancerous thyroid tissue proving a reliable tumor marker to detect malignant

transformation of thyroid cancer. A study done by Saussez et al shows that 87% of

macro-papillary carcinomas and 67% of micro-papillary carcinomas had detectable

serum galectin-3. This study, including 71 patients with multiple thyroid nodules and 13

with a single thyroid nodule, indicated that the serum galectin-3 test was more sensitive

with multiple thyroid nodules detecting 74 % of the papillary tumor carcinomas in the

group with multiple nodules and only 11% of the papillary tumor carcinomas in the group

with single thyroid nodules.14

The presence of galectin-3 in thyroid nodules and other head and neck cancers has

also been correlated with the aggressiveness of the cancer.15, 16

Savin et al immunostained

for galectin-3 in combination with thyroid peroxidase in differentiated thyroid

carcinomas to evaluate if the expression of galectin-3 correlates to the aggressiveness of

thyroid cancer. They found that in the papillary carcinoma there was high galectin-3

expression with increased tumor size, nodal involvement, tumor invasion outside the

7

thyroid and metastasis. Galectin-3 levels are also corollary to the extent of invasiveness

of follicular thyroid carcinoma.16

In 1991, Lotan et al determined that there was increased content of two lactose

binding lectins in human colorectal cancers that had progressed to metastasis; one of

those is now known to be galectin-3. In Lotan‟s studies, the concentration of galectin-3

was greater in tumors in the advanced cancer stage, “Dukes stage D”.17

Lacovazzi et al

studied galectin-3 levels in colorectal cancer and also concluded that galectin-3 levels

were higher in more differentiated tumors.18

In rat models, induced tumors were

evaluated for the presence of galectin-3 during colon carcinogenesis. The normal colon

cells in the untreated rats showed no staining for galectin-3. The neoplastic colon cells

were strongly stained for galectin-3 in the early stages, showing that galectin-3 may be

involved in the early stages of neoplastic transformation of colon cancer.19

The expression of galectin-3 in gastric tumors was analyzed in the early 1990‟s.

Lotan evaluated normal mucosa, primary gastric carcinomas and metastatic gastric

carcinomas for the expression of galectin-3 via immunoblotting. The primary tumors in 9

out of 26 patients showed an increase in galectin-3 expression compared to the patients‟

normal mucosa. However, galectin-3 levels were close to the same in 14 out of 26

patients, and in three of the cases there was less galectin-3 expressed in the primary

tumor compared to the normal mucosa.20

In about half of the tumors the level of

expression of galectin-3 was significantly higher in the primary tumor compared with the

metastatic tumor cells. However, galectin-3 expression levels in liver and lymph

metastases were higher than expression levels in primary tumors. The conclusion was

that galectin-3 is implicated in the metastatic phenotype but is not sufficient to predict the

http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A(Iacovazzi%252C%2BPalma%2BA.)



8

metastatic tendency of a primary tumor.20

More recent studies reveal that galectin-3

increases tumor cell motility in malignant gastric tissues containing high concentrations

of galectin-3.21

Saussez et al investigated the upregulation of galectin-3 during tumor progression

in head and neck cancer. Immunohistochemistry was used to quantitatively determine the

amount of galectin-3. A polyclonal antibody was used against galectin-3. They evaluated

a series of 79 hypopharyngeal squamous cell carcinomas (HSCC) compared with 16

epithelia with varying levels of dysplasia. In addition, a series of 58 laryngeal squamous

cell carcinomas (LSCC) was compared with 34 epithelia with varying levels of dysplasia.

The galectin-3 levels in the neoplastic HSCC and LSCC cells were significantly higher

than any of the epithelial cells. The data shows a correlation of galectin-3 expression and

neoplastic progression of both HSCC and LSCC.22

In summary, galectin-3 is over-expressed in several types of cancer. This makes

the protein a good target for the investigation of cancer progression.

Galectin-3 in Metastasis

Metastatic spread of tumor cells is responsible for 90% of cancer deaths.23

In

certain cancers, galectin-3 expression has been correlated with the metastatic potential.

Evidence suggests that galectin-3 plays a key role in the metastasis of several

carcinomas.13

The metastatic cascade is a multistep process requiring numerous cell to cell and

cell to extracellular matrix (ECM) interactions. These steps include: primary tumor

formation, angiogenesis, localized invasion and intravasation, transport through

9

circulatory system, arrest in micro vessels, extravasation, formation of micro-metastasis

and colonization.23

Galectin-3 has a role in several of these tumor progression steps.

Primary Tumor Formation:

Galectin-3 in Homotypic Cellular Aggregation

Cancer studies reveal that changes in homotypic and heterotypic cellular adhesion

are vital initial steps in cancer progression and lead to metastasis. Extracellular galectins

are known to be involved in the homotypic and heterotypic aggregation processes and

therefore are good candidates for the study of tumor formation, adhesion, and

metastasis.24

Galectins bind to cell-surface glycoconjugates to serve several biological roles.

The galectins can trigger signaling events within the cell and can cross-link cell-surface

glycoconjugates. This cross-linking can induce bridging of tumor cells to each other

leading to tumor formations; this is known as homotypic aggregation. Galectins can also

bridge tumor cells with other components within the extracellular matrix, such as

laminin, collagen, asialofetuin, fibronectin, vitronectin, or endothelial cells causing

heterotypic aggregation.25, 26

Studies from the Karmanos Cancer Institute at Wayne State University were some

of the first to provide evidence that galectin-3 may play a role in tumor cell embolization.

Dr. Raz et al studied galectin-3 and one of its natural ligands, the heavily N-glycosylated

protein, Mac-2 binding protein (BP). In the presence of Mac-2-BP, A375 human

melanoma cells with high expression of galectin-3 formed homotypic aggregates. This

aggregation was inhibited in the presence of the competitive galectin-3 inhibitors lactose

and anti-galectin-3 antibody.27

10

Previous studies have shown that cell surface galectin-3 is involved in

asialofetuin-induced homotypic aggregation. For example, galectin-3 was expressed on

the cell surface of Sf9 cells, which do not express galectin-3 or aggregate naturally. In the

presence of exogenous glycoprotein (i.e. asialofetuin) the new galectin-3 infected Sf9

cells underwent homotypic aggregation. Further, lactose and anti-galectin-3 antibodies

inhibited the aggregation. This study demonstrated that cell surface galectin-3 is involved

in mediating glycoprotein-induced homotypic aggregation.28

Galectin-3 in Angiogenesis

Another key process in tumor progression, and a precursor to metastasis, is

carbohydrate-dependent endothelial cell morphogenesis leading to angiogenesis. Tumor

angiogenesis requires the recruitment of endothelial cells to form blood vessels. Galectin-

3 serves as a chemoattractant of endothelial cells.29

The binding of extracellular galectin-3 to endothelial cells has been shown to

affect capillary tube formation in vitro and angiogenesis in vivo. To study the effect of

galectin-3 on endothelial cell tube formation, a dose response of HUVEC-C to soluble

human galectin-3 was analyzed by Raz et al. Varying concentrations of galectin-3 were

added to HUVEC-C cells plated on a gel formed by diluted Matrigel. Increased galectin-

3 enhanced endothelial cell organization. To further show that this galectin-3 interaction

is mediated by its carbohydrate-binding domain, the HUVECs were plated on a Matrigel

gel in the presence of competitive saccharides, modified citrus pectin (MCP) and lactose.

The cellular organization was completely inhibited by MCP and partially inhibited by

lactose.29

11

To further evaluate the involvement of galectin-3 in angiogenesis, Raz et al

injected galectin-3 expressing and non-expressing tumors into nude mice. The tumors

were evaluated for the presence of blood vessels. The galectin-3 expressing tumor

contained blood vessels, while the galectin-3 negative tumor had no blood vessels.29

Galectin-3 also stimulates motility of endothelial cells. Nangia-Makker et al

performed chemotaxis assays in which the endothelial cells showed a dose-dependent

chemotactic response to galectin-3 and conditioned medium of cells secreting

galectin-3.29

Localized Invasion, Intravasation and

Extravasation: Galectin-3 in Heterotypic Cellular Aggregation

Cancer cells must leave the primary tumor site through the extracellular matrix,

the basement membrane and endothelial cells lining the blood vessels, intravasation,

enter the circulatory system and finally leave the blood vessels and re-enter into a new

tissue, extravasation, to form micro-metastases.23

Intravasation and extravasation require

a change in the adhesion properties of the cells and significant interaction with the

extracellular matrix and basement membrane.30

Galectin-3 is involved in the binding to

carbohydrates present on glycoproteins, glycolipids and glycosaminoglycans in the

extracellular matrix and the endothelial cell surface to mediate interactions of the

invading or circulating cancer cells.13

Several studies have confirmed that transfection of galectin-3 into cells increased

cell adhesion to components of the extracellular matrix, such as laminin, and increased

tumor associated integrin expression.25, 31

12

An endothelial cell heteroypic adhesion study was performed on highly metastatic

human breast carcinoma cells, MDA-MB-435 and its non-metastatic counterpart, MDA-

MB-468. The MDA-MB-435 line expresses high levels of galectin-3 and Tf antigen,

whereas the non-metastatic MDA-MB-468 has much lower levels of galectin-3 and Tf

antigen. Metastatic MDA-MB-435 had increased adhesion to monolayers of endothelial

cells and after attachment induced homotypic aggregation creating multi-cellular

aggregates. The galectin-3 was observed to be localized near the heterotypic and

homotypic contact sites using confocal microscopy and fluorescence-activated cell sorter

analysis. These results imply that galectin-3 together with Tf antigen is involved in

cellular adhesion processes to the endothelial blood vessel lining.26

Formation of Micro Metastasis and Colonization:

Galectin-3 in Embolism and Secondary Tumor Formation

Secondary tumors form in a similar manner as primary tumors. The circulating

neoplastic cells from the original primary site gain ability to adhere with other tumor cells

and nearby tissue cells. The cancer cells form small aggregates within blood vessels

creating a block, or embolus, in the micro-capillaries. The embolus then extravasates at

the secondary site and the cancer cells colonize.13

The ability of galectin-3 to homotypically and heterotypically aggregate is a key

factor in secondary tumor formation just as it is in primary tumor formation. Platt et al

used modified citrus pectin (MCP), a galactoside competitive galectin-3 inhibitor

containing simple sugars, to determine its affect on B16-F1 melanoma tumor lung

colonization in vivo in mice. The mice given MCP had a greater than 90% decrease in

lung colonization of the B16-F1 cells.32

13

The cell surface galectin-3 enhances the adherence of cancer cells to the ECM

through its carbohydrate-binding activity. This lectin/ECM

interaction in turn mediates

tumor cell to cell interactions causing aggregation leading to emboli formation, a

precursor to metastasis.25

Summary of Galectin-3 in Cancer

Expression of galectin-3 has been shown to correlate with tumor progression and

metastatic potential of many cancers. Galectin-3 binds to cell surface carbohydrates.

These adhering properties, along with the ability to cross-link leads to many roles for

galectin-3 in neoplastic transformation including: homotypic and heterotypic aggregation

leading to primary and metastatic tumor formation, invasion, intravasation and

extravasation; angiogenesis, and metastasis.

As a key component in tumor progression, galectin-3 has become an obvious

target for cancer studies. The next chapter will discuss the synthetic designs used for

studying galectin-3, its interactions with carbohydrates and its effects on cancer cells.

14

CHAPTER 3

CARBOHYDRATE FUNCTIONALIZED SYSTEMS TO STUDY GALECTIN-3

Introduction to Carbohydrate Functionalized Systems

As described in the previous chapter, galectin-3 is a critical participant in cancer

progression. Galectin-3 interacts with abundantly available carbohydrates to enhance

homotypic and heterotypic aggregation, angiogenesis, invasion, intravasation and

extravasation of cancer cells. Fully elucidating the behavior of galectin-3, and galectins in

general, will advance understanding of many cancer cell lines and may lead to the

development of novel therapeutic agents. Carbohydrate-functionalized systems are

currently being used to study galectin-3/carbohydrate interactions and to provide

inhibitors for the tumorigenic protein. The goal of this project is to study galectin-

3/carbohydrate interactions at the cellular level as they pertain to tumor growth and

metastasis using synthetically-produced carbohydrate-functionalized dendrimers.

Carbohydrate-Functionalized Systems to Study Galectin-3/Carbohydrate Interactions

Multivalent interactions are very common and important in biological

mechanisms. Monovalent lectin/carbohydrate interactions are very weak with milli- to

micro-molar dissociation constants. Multivalent interactions enhance this binding by use

of multiple receptors to bind several ligands. Many molecules in nature have a specific

multivalent, “dendritic” architecture to increase weak monovalent binding.2 A variety of

carbohydrate architectures have been developed to study multivalent carbohydrate/

protein interactions, such as galectin-3 interactions with its respective ligands. These

15

multivalent interactions with galectin-3 can be used to form galectin-3/glycodendrimer

clusters which will effectively mediate cellular aggregation. Traditional small-molecule

therapeutics cannot form these novel matrix architectures created through the larger

glycodendrimer.

Modified citrus pectin (MCP) has been used to competitively block the galectin-3

carbohydrate-binding domain. MCP is the hydrolyzed form of citrus pectin, a highly

complex branched polysaccharide fiber rich in galactoside residues present in all citrus

fruit. Hydrolysis of citrus pectin produces smaller, linear, water-soluble MCP fibers that

act as a galectin-3 ligand. Human breast carcinoma cells (MDA-MB-435) were injected

in the mammary fat pad region of MCP-fed nude mice. The tumor volume in the MCP-

treated mice was significantly reduced compared to the control group (Figure 3.1).33

The

multivalent carbohydrate, MCP, competitively blocks the galectin-3 carbohydrate-

binding domain to prevent cell surface galectin-3 from binding to its natural carbohydrate

ligands, inhibiting tumor progression and metastasis. This study, along with a handful of

studies utilizing modified citrus pectin, suggests the potential for carbohydrate-mediated

cancer therapy.32

34

35

36

37

38

39

40

41

Figure 3.1. Tumor growth reduction in mice treated with modified citrus pectin.32,

33

16

Figure 3.2 shows the results for a study investigating a variety of oligosaccharide

derivatives (Figure 3.3) investigated to “mimic the endogenous ligands for different

lectins, thus affecting survival, adhesion and migration of normal or neoplastic cells.”

The analogs were characterized for galectin-1 and -3 inhibition through a homotypic

aggregation assay with A375 human melanoma cells and tumor cell apoptosis evaluation

(not shown). Results revealed that oligosaccharides 2, 3 and 11 were among the most

effective inhibitors of the galectins. The most potent inhibitor of the oligosaccharide

derivatives was the allyl lactoside 3.24

Figure 3.2. The effect of oligosaccaride derivates on galectin-3

induced homotypic aggregation of A375 cells.24

17

Figure 3.3. Structures of oligosaccharide derived galectin inhibitors.24

Andre´et al synthesized di-, tri- and tetravalent lactoside-bearing glycoclusters

with an affinity to lectins. The affinities of the glycoclusters to lectins were assessed in a

competitive solid-phase binding assay with labeled sugar receptors, including galectin-3.

Results show that the trivalent lactocluster (shown in Figure 3.4) surpassed the inhibitory

capacity of lactose. The trivalent cluster was found to have an IC50 value of 30.8 mM

compared to a 700 mM IC50 for free lactose.42

18

Figure 3.4. Tri-valent rigidified lactoside clusters tested for lectin inhibition. 42

Gold nanoparticles bearing the Thomsen–Friedenreich disaccharide were

developed to evaluate multivalent protein/carbohydrate interactions by Barchi et al. The

Thomsen-Fiedenreich (TF) antigen is known to be over-expressed on the surface of

tumor cells and is believed to be involved in tumor progression, including invasion and

metastasis of some cancers, specifically, breast and prostate cancer.43

As mentioned in

chapter two, the TF antigen is a ligand to galectin-3. Galectin-3 cross-links tumor cells to

the endothelium through the carbohydrate binding of TF antigen. Barchi et al

synthesized the “gold nanoshells encapsulated with up to 90 units of the (TF) tumor-

associated carbohydrate antigen (TACA) disaccharide (Galb1-3GalNAc-α-O-Ser/Thr) as

well as the assembly of a suitably linked designer glycopeptide as a precursor to similar

multivalent presentations on gold,” in hopes that their carbohydrate scaffold may “lead to

the development of therapeutic agents that inhibit protein–carbohydrate interactions.”44

Stoddart et al developed a lactoside-displaying cyclodextrin (CD)

pseudopolyrotaxane to test its ability to inhibit the aggregation ability of galectin-1. The

19

CDs are able to alter the display of the lactoside ligand via rotation around the axis of the

polymer chain and translational movement along a “linear polyviologen string,” (Figure

3.5) giving the pseudopolyrotaxane dynamic, multivalent binding capabilities.45

Figure 3.5. The pseudopolyrotaxane. The backbone polyviologen string is blue.

The polymer chain axis is green. The lactoside-displaying cyclodextrin

is red.44

These studies explored multivalent carbohydrate interactions with the use of

synthetic carbohydrate systems. Each carbohydrate scaffold explored lacks a readily

controlled, systematic manipulation. The results indicate that a systematic scaffold to

study galectin/carbohydrate interactions such as the dendrimer described in the following

section is warranted.

The Dendrimer Framework

The goal of this project is to study galectin-3/carbohydrate interactions at the

cellular level as they pertain to tumor growth and metastasis using synthetically-produced

carbohydrate-functionalized dendrimers. The dendrimers serve as an excellent platform

for the display of carbohydrates and for the study of multivalent protein-carbohydrate

interactions due to their ease of size manipulation and their straightforward functional

20

group modification. The glycodendrimers have been synthesized to interfere with

galectin-3/carbohydrate interactions that induce tumor progression and metastasis (Figure

3.6 (a) & (b)).

(a) (b)

Figure 3.6. (a) A schematic representation of a dendrimer interacting with the active site

of galectin-3 and inhibiting cellular aggregation. (b) A schematic representation of a

dendrimer interacting with multiple galectin-3 proteins and promoting aggregation.

Polyamidoamine (PAMAM) dendrimers are macromolecules that are uniformly

branched around an inner core.46

PAMAM dendrimers range from the smaller generation

zero, G(0), with only four branches with surface groups to which carbohydrates can be

attached, to generation ten, G(10), with 4,096 surface groups (Figure 3.7). The

dendrimers used in this research, generations 2, 3, 4 and 6, were purchased from

Dendritech, Inc. See Table 3.1 for the calculated properties of G(0) – G(6) PAMAM

dendrimers from Dendritech, Inc. 47

21

(a) (b)

N N NH

O

HN O

HN

O

N

HN

O

NH

O

NNHN

HN O

O

NH2

H2N

NHO

HN

O

NH2

HN O

N

HN

O

NH

O

NNNH

HN O

O

NH2

H2N

NHO

NH

O

NH2

NH2

N

NHO

HN

O

N

NH

O

HN

O

NH2

N

NHO

NH

O

NH2

NH2

N

HNO

NH

O

N

HNONH

O

H2N

N

HN OHN

O

H2N

NH2

H2N

NH2

NH2

G(0) = blue

G(1) = red

G(2) = purple

H2NCH2CH2NH2

(B) H2NCH2CH2NH2

(A,B) (A,B)(A) CH2=CHCO2Me

Figure 3.7. (a) PAMAM dendrimer scaffold generations 0 (blue), 1 (red) and 2 (purple).

(b) Three dimensional representations of the “tree like” branching PAMAM dendrimer.

Table 3.1. Theoretical calculated properties of amine surface functional PAMAM

dendrimers by generation. 47

Generation Molecular

Weight

(Daltons)

Measured

Diameter (Å)

Surface

Groups

0 517 15 4

1 1,430 22 8

2 3,256 29 16

3 6,909 36 32

4 14,215 45 64

5 28,826 54 128

6 58,048 67 256

In nature, carbohydrate interactions are often multivalent. The dendrimer is a

useful platform for biologically relevant multivalent carbohydrate/ligand interactions

since it is well-defined three dimensionally and has an increasing number of branches

with each increasing generation that can be functionalized with carbohydrates to bind into

multiple lectin binding sites.48

22

The PAMAM dendrimers have amino surface groups that are readily

functionalized to make them biologically relevant, serving as a distinctive framework to

study galectin-3 interactions with carbohydrates. For this study, dendrimers of

generations 2, 3, 4, and 6 were functionalized with the β-galactosides lactose, galactose,

and N-acetyllactosamine (LacNAc) (Figure 3.8).49

(a)

(b)

(c)

Figure 3.8. Functionalized dendrimers. (a) Lactose functionalized dendrimers:

generation 2, 1; generation 3, 2; generation 4, 3; and generation 6, 4. (b)

Galactose functionalized dendrimers: generation 3, 5; generation 4, 6; and

generation 6, 7. (c) LacNAc functionalized dendrimer generation 4, 8. 49

23

Generation 2 lactose, 1, has a weighted average molecular weight (Mw) of

9,383 g/mol. 1 is functionalized with sixteen lactose sugars. Generation 3 lactose, 2 (Mw

= 15,000 g/mol), contains 23 lactose sugars. Generation 4 lactose, 3 (Mw = 31,200

g/mol), contains 54 lactose sugars. Generation 6 lactose, 4 (Mw = 100,000 g/mol),

contains 125 lactose sugars. Generation 3 galactose, 5 (Mw = 15,200 g/mol), contains 26

galactose sugars. Generation 4 galactose, 6 (Mw = 31,500), contains 57 galactose sugars.

Generation 6 galactose, 7 (Mw = 101,000), contains 145 lactose sugars. Generation 4

LacNAc, 8 (Mw = 32,035 g/mol), contains 50 lactose sugars.

Summary of Carbohydrate Functionalized Systems to Study Galectin-3

Galectin-3 interactions with carbohydrates are significant in tumor progression and

metastasis. The objective in this project is to study these protein/carbohydrate interactions

and possibly to develop a therapeutic agent to interfere with cancer progressing processes

using carbohydrate-functionalized dendrimers.

To date, results have been obtained using a spectrum of glycodendrimers, 1-8, in a

homotypic aggregation cell based assay that mimics tumor formation through direct

carbohydrate/galectin-3 interactions. Results confirm glycodendrimer interaction and

mediation of galectin-3/carbohydrate interactions.

24

CHAPTER 4

HOMOTYPIC AGGREGATION STUDIES

Background

In vitro cell-based assays are an important precursor to in vivo studies. Since live

cells are used, cell-based assays offer a limited, accurate representation of live-models.50

Several studies have successfully used homotypic aggregation assays to evaluate cell

behavior.24,32,44,50

Raz et al showed, using a homotypic aggregation assay, that tumor galactoside-

binding proteins such as galectin-3 mediate cellular recognition. They demonstrated this

by competitively interfering with the cross-linking of carbohydrates on adjacent cells in

the homotypic aggregation assay with modified citrus pectin.32

Iurisci et al, Zou et al and Stoddart et al all successfully used homotypic

aggregation studies to test synthetic inhibitors of galectins as described in Chapter 3.24,51,45

Goals

In this project, the homotypic aggregation cell-based assay was chosen to screen a

number of glycodendrimers in less time than could be achieved in any comparable in vivo

study.

A homotypic aggregation assay was chosen to study galectin-3/carbohydrate

interactions at the cellular level as they pertain to tumor growth and metastasis with the

use of synthetically-produced carbohydrate-functionalized dendrimers. The assays were

performed as a first step in evaluating the effects of glycodendrimers in galectin-3

25

influenced carcinomas by mediating lectin induced cancer cell homotypic aggregation via

interactions with the carbohydrate-functionalized dendrimers.

Project Description

Galectin-3 expressed on the cell surface is involved in homotypic aggregation of

cancer cells contributing to tumor aggregation and tumor cell emboli formation.28

In this

research, the effect of the functionalized dendrimers on cell-cell interactions mediated by

galectin-3 is studied through in vitro cell-based homotypic aggregation assays. The

hypothesis is that carbohydrate-functionalized dendrimers bearing varying β-galactosides

will mimic natural galectin-3 ligands and affect cancer cell-cell aggregation.

To determine whether cell-cell aggregation is influenced by glycodendrimers,

confluent HT-1080 human fibrosarcoma cells from ATCC were harvested using 2 mM

EDTA. Cells were suspended in a mixture of serum free media, galectin-3, and

glycosystem. Varying concentrations of glycodendrimer (0 – 20 µL of 2 mg/mL stock

solution) were used. A constant amount of galectin-3 such that the final concentration of

galectin-3 per sample was 120 µg/mL was combined with the glycodendrimer in an

eppendorf tube. Cells were suspended in serum free media so that 240,000 cells were

added per sample and a total of 50 µL of solution was present per tube. This mixture was

vortexed to mix the compounds. After 5 minutes of incubation at 37 °C on a rotator,

videos were taken of the cells from the eppendorf tubes at 10x magnification on a

microscope. Videos were also taken after one hour of incubation. Single cells were

counted and the following equation was used to calculate the extent of aggregation:

1-(Nt/Nc) ×100, where Nt represents the number of single cells in the test suspension

26

which is in the presence of dendrimer and Nc represents the number of single cells in the

control which is in the absence of dendrimer.24

Figure 4.1 shows the procedural steps.

Figure 4.1. Homotypic aggregation assay procedure.

Several control assays were performed (Figure 4.2). Each experiment had a

negative and positive control. The negative control was the HT-1080 cells alone. The

positive control was HT-1080 plus a final concentration of 120 μg/mL galectin-3.

Glycodendrimers were also added to HT-1080 cells without galectin-3. The last control

was the addition of galectin-3 with a truncated N-terminal domain (CRD galectin-3).

27

Figure 4.2. Control homotypic aggregation assay cell pictures. (a) HT-1080 cells

alone: representative picture of the negative control with little to no aggregation.

(b) HT-1080 cells plus 120 µg/mL final concentration of galectin-3:

representative picture of the positive control with aggregation. (c) HT-1080 cells

plus dendrimer. No aggregation is seen in this control. (d) HT-1080 cells plus

galectin-3 CRD without N-terminal domain. No aggregation is seen in this

control.

Results indicating that varying generations of dendrimer alter the effect on cancer

cell homotypic aggregation are described in the following section. Also, different

carbohydrate surface groups on the dendrimer have varying effects on the homotypic

aggregation.

Results

A variety of potential inhibitors have been previously developed to prevent the

known aggregation action of galectin-3. Here, multivalent dendrimers 1-8 were

functionalized with carbohydrates and were screened to determine the effect they have on

28

galectin-3 induced cancer cell aggregation. As seen in Figures 4.3-4.24, varying

carbohydrates on the dendrimers have different effects in the homotypic aggregation.

Varying generations of dendrimer also vary the effect on the tumor cell aggregation.

The control assays seen in Figure 4.2 provide the standard for the results

presented in this research. The negative control in each experiment is the standard 0%

aggregation from which the rest of the cell solutions were compared. The positive control

is the standard 100% aggregation from which the results were compared. Addition of

glycodendrimers and CRD galectin-3 to HT-1080 cells did not affect cellular

aggregation, confirming that the galectin-3 is necessary to induce aggregation.

Cancer cell aggregation was induced with the addition of galectin-3. To determine

that galectin-3 alone caused the aggregation, a control assay was performed. Lactose, a

ligand of galectin-3, was added to the galectin-3 before the cells were added. Increased

inhibition of tumor cell aggregation correlated with an increased concentration of lactose,

confirming galectin-3 as the inducer of aggregation (Figures 4.3 & 4.4).

29

Lactose

Figure 4.3. The effects of increasing concentrations of lactose on galectin-3 induced homotypic HT-1080

aggregation. As the concentration of lactose increases the aggregation of HT-1080 cells decreases.

Figure 4.4. Pictures of HT-1080 cellular aggregation with galectin-3 and lactose. A) Negative control: HT-

1080 cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and

701 µM lactose. D) HT-1080 cells with galectin-3 and 1,420 µM lactose. E) HT-1080 cells with galectin-3

and 2,337 µM lactose.

30

O O

HO

OH

O

OH

O

HN

G(2) PAMAM

OH

O

HO

OH

OH

HN

S16

1

Figure 4.5. The effects of increasing concentrations of functionalized dendrimer 1 on galectin-3 induced

homotypic HT-1080 aggregation. As the concentration of 1 increases the aggregation of HT-1080 cells

decreases.

Figure 4.6. Pictures of HT-1080 cellular aggregation with galectin-3 and 1. A) Negative control: HT-1080

cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 85.3

µM 1.

31

O O

HO

OH

O

OH

O

HN

G(2) PAMAM

OH

O

HO

OH

OH

HN

S16

1


homotypic HT-1080 aggregation. There is a significant decrease in HT-1080 aggregation at 51.2 µM of 1.



µM 1. D) HT-1080 cells with galectin-3 and 34.1 µM 1. E) HT-1080 cells with galectin-3 and 51.2 µM 1.

32

O O

HO

OH

O

OH

O

HN

G(2) PAMAM

OH

O

HO

OH

OH

HN

S16

1

Figure 4.9 The effects of increasing concentrations of functionalized dendrimer 1 on galectin-3 induced

homotypic HT-1080 aggregation. As the concentration of 1 increases the aggregation of HT-1080 cells decreases.




F) HT-1080 cells with galectin-3 and 51.2 µM 1.

33

O O

HO

OH

O

OH

O

HN

G(2) PAMAM

OH

O

HO

OH

OH

HN

S16

1


homotypic HT-1080 aggregation. There is a decrease in HT-1080 aggregation at 51.2 µM of 1.



µM 1. D) HT-1080 cells with galectin-3 and 51.2 µM 1.

34

O O

HO

OH

O

OH

O

HN

G(3) PAMAM

OH

O

HO

OH

OH

HN

S27

2


homotypic HT-1080 aggregation. There is a decrease in HT-1080aggregation at 26.7 µM of 2.

Figure 4.14 Pictures of HT-1080 cellular aggregation with galectin-3 and 2. A) Negative control: HT-1080



35

O O

HO

OH

O

OH

O

HN

G(3) PAMAM

OH

O

HO

OH

OH

HN

S27

2


homotypic HT-1080 aggregation. There is a decrease in HT-1080 aggregation at 26.7 µM of 2.



µM 2. D) HT-1080 cells with galectin-3 and 16.0 µM 2. E) HT-1080 cells with galectin-3 and 26.7 µM 2

F) HT-1080 cells with galectin-3 and 32.0 µM 2.

36

O O

HO

OH

O

OH

O

HN

G(3) PAMAM

OH

O

HO

OH

OH

HN

S27

2

Figure 4.17. The effect of 32.0 µM of functionalized dendrimer 2 on galectin-3 induced homotypic HT-

1080 aggregation. There is a significant decrease in HT-1080 aggregation.



µM 2.

37

O O

HO

OH

O

OH

O

HN

G(6) PAMAM

OH

O

HO

OH

OH

HN

S 148

4

Figure 4.19. The effects of increasing concentration of functionalized dendrimer 4 on galectin-3 induced

homotypic HT-1080 aggregation. There is a no significant change in HT-1080 aggregation.



µM 4. D) HT-1080 cells with galectin-3 and 6.4 µM 4.

38

HO

O

HO

OH

O

OH

O

HN

G(3) PAMAM

HN

S 26

5


homotypic HT-1080 aggregation. There is a no significant change in HT-1080 aggregation.




39

O O

HO

NHAc

O

OH

O

HN

G(4) PAMAM

OH

O

HO

OH

OH

HN

S 27 8


homotypic HT-1080 aggregation. There is an increase in HT-1080 aggregation with an increase in concentration of 8.

Figure 4.24. Pictures of HT-1080 cellular aggregation with galectin-3 and 8. A) Negative control: HT-1080 cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 15.0

µM 8.

40

Analysis

Homotypic aggregation studies were performed on HT-1080 cells. Aggregation of

the cells was induced by galectin-3 and varying generations and types of glycodendrimers

were analyzed for their effects on the aggregation. The data was represented graphically

as the % aggregation of HT-1080 cells as a function of glycodendrimer concentration.

The percent aggregation was calculated using the following equation: 1-(Nt/Nc) x 100.

The pictures are representative still images of live video taken of the cells through a

microscope and 10x magnification. The homotypic aggregation results acquired indicate

that the carbohydrate-functionalized dendrimers have an effect on cancer cell

aggregation.

The overall trends of glycodendrimers 1-8 in the homotypic aggregation assays

are as follows. Lactose causes a decrease in cellular aggregation with an increase in

concentration. 1 shows a significant decrease of aggregation with increasing

concentrations whereas 2 has the same general trend but is not as consistent. Compound 3

gives less consistent results than 1or 2. The results for 3 were not readily reproducible.

Compounds 4, 5, 6, and 7 don‟t have a notable effect on the aggregation of HT-1080

cells. Glycodendrimer 8 increases the tumor cell aggregation.

Glycodendrimers 1 and 2 inhibited aggregation of HT-1080 cancer cells induced

by galectin-3 (Figures 4.7-4.18). Inhibition trends are shown in Table 4.1. Compound 1

significantly inhibited tumor cell aggregation. Overall, 100% inhibition was observed

with 1 at a concentration of 51.2 µM of dendrimer and greater whereas 100% inhibition

was not attained with lactose. Glycodendrimer 1 inhibited 50% or greater of cellular

41

aggregation at concentrations of 8.5 µM – 51.2 µM whereas 2,337 µM of lactose was

required to inhibit at least 50% of aggregation. Glycodendrimer concentrations of 1 that

correlate with at least a 50% or greater inhibition have a 46 – 275 fold increase in

inhibition compared to lactose. This is a 2.8 – 17 fold increase in inhibition capacity per

lactose sugar considering 1 contains 16 lactose units.

Glycodendrimer 2 also inhibited galectin-3 induced cellular aggregation. Overall,

nearly 100% inhibition was observed with 2 at a concentration of 32.0 µM in one noted

assay (see Figures 4.17 & 4.18). Glycodendrimer 2 inhibited 50% or greater of cellular

aggregation at concentrations of 26.7 µM & 32 µM whereas 2,337 µM of lactose was

required to inhibit at least 50% of aggregation. Glycodendrimer concentrations of 2 that

correlate with at least a 50% or greater inhibition have a 73-88 fold increase in inhibition

compared to lactose. This is a 3.2 – 3.8 fold increase in inhibition capacity per lactose

sugar since 2 contains 23 lactose units.

Table 4.1. Concentrations of inhibitory compounds.

Compound

(sugar units

per

compound)

≥ 50%

inhibition

(µM/

compound)

≥ 50%

inhibition

(µM/

sugar)

≈ 100%

inhibition

(µM/

compound)

≈ 100%

inhibition

(µM/

sugar)

x fold

enhancement per

compound of ≥ 50% inhibition

compared to lactose

x fold

enhancement

per sugar of ≥ 50% inhibition

compared to lactose

Lactose (1) 2,337 2,337 - - - -

1(16) 8.5 136 51.2 819 275 17

1 (16) 42.6 682 51.2 819 55 3.4

1 (16) 51.2 819 51.2 819 46 2.8

2 (23) 26.7 613 - - 88 3.8

2 (23) 32.0 736 32.0 736 73 3.2

Lactose functionalized dendrimers 3 & 4 varied significantly in their mediation of

cellular aggregation. Neither compound afforded consistant results for the numerous

assays that were performed.

42

Galactose functionalized dendrimers 5-7 of all generations were all found to have

little to no effect on galectin-3 induced cellular aggregation. Results for studies using

galactose functionalized G(3) are shown in Figures 4.21 & 4.22.

Dendrimer functionalized with LacNAc, 8, increased cellular homotypic aggregation

up to 134% relative to the control galectin-3 induced aggregation at a concentration of 10

μM of dendrimer. (Figures 4.23 & 4.24)

Both inhibitory compounds 1 and 2 are functionalized with lactose. The

glycodendrimers that did not have an effect on HT-1080 aggregation were functionalized

with galactose. The compound that enhanced cellular aggregation presented LacNAc

saccharides. The monovalent binding affinity of galectin-3 for lactose is about 8-times

greater than that of galactose. LacNAc binds galectin-3 up to 7.5-fold better than lactose

binds galectin-3.11

These binding affinities, along with the size (generation) of dendrimer,

may explain the results acquired through the homotypic aggregation assays.

It is proposed that the differences in galectin-3 binding affinities for the

saccharides alters the galectin/glycodendrimer clustering which influences the exposure

of the galectin-3 CRD for binding to the tumor cell surface carbohydrates. With a low

binding affinity for galactose, it is thought that the galectin-3 only had slight interaction

with or no interaction at all with the galactose carbohydrates on the dendrimer

(compounds 5-7), therefore the galactose functionalized dendrimers did not create a

matrix that would manipulate the cellular aggregation. Galectin-3 has a much higher

affinity for lactose and LacNAc, therefore the interactions between the galectin and these

glycodendrimers are significant and create a mediating environment by blocking galectin-

3 binding to native cell surface carbohydrates.

43

It is possible that the pattern of galectin-3 that is displayed to the cells is altered

by the glycodendrimers. The size (generation) of the dendrimer seems to be a

determining factor of this pattern, influencing inhibition or enhancement of aggregation.

Figure 4.25 is a schematic representation of what may be occuring with the different

sized dendrimers. Figure 4.25 A shows the smaller generation 2 and 3 dendrimers 1 and 2

sequestering the binding site of galectin-3, competitively inhibiting the galectin-3

interactions with saccharides present on the cell surface and preventing galectin-3

bridging of cells. Figure 4.25 (b) shows a schematic representation of what may occur

with the larger generations of dendrimers. Several galectins can bind around the larger

dendrimer, creating a scaffold conducive to enhanced aggregation.

(a) (b)

Figure 4.25 (a) A schematic representation of the smaller generations of functionalized-

dendrimers inhibiting cellular aggregation. (b) A schematic representation of the larger

generations of functionalized-dendrimers enhancing and varying cellular aggregation.

Dendrimers are an improvement on the other carbohydrate-recognition systems

discussed previously. Glycodendrimers more readily lend themselves to the following

significant composite design functions: easy control of size, systematic studies,

multivalent properties, and numerous amine end groups that can be easily functionalized

with multiple biologically relevant end groups.

44

CHAPTER 5

SUMMARY, CONCLUSIONS AND FUTURE WORK

Chimeric galectin-3, in the galectin family of proteins that bind β-galactosides, is

a diverse protein that has several biological functions. The cellular adhesion properties of

galectin-3 are particularly important to the research presented here. Galectin-3 binds and

cross-links cell surface carbohydrates. In normal cells, galectin-3 regulates cellular

adhesion. In neoplastic cells galectin-3 induces homotypic and heterotypic aggregation,

facilitating tumor formation.

Galectin-3 has differential levels of expression in cancer cells. In many cancers,

neoplastic progression to metastasis has been correlated with increasing levels of

galectin-3. This protein has been implicated in several tumorigenic roles including

primary and secondary tumor formation, angiogenesis and chemotaxis, localized

invasion, intravasation and extravasation and metastasis. The increased levels of galectin-

3 in cancer cells and its many functions in cancer progression make it a good target for

the study of cancer and possible novel therapeutics.

Many studies to date have probed the multivalent carbohydrate interactions of

galectin-3 with the use of synthetic carbohydrate systems. These systems are developed

to interfere with galectin-3 binding to aberrantly expressed carbohydrates in tumor cells.

The results of previous experiment indicate that a systematic scaffold to study

galectin/carbohydrate interactions such as the one described here is warranted.

In the described research, the synthetic carbohydrate system has been refined

from previous experiments. The PAMAM dendrimer is an ideal scaffold for the display

45

of appropriate carbohydrates. The tree-like branching structure offers the multivalency

that exists in the natural system. Dendrimers are also easy to manipulate for any given

situation. They range in size from generation 0 to 10 and can be functionalized with a

variety of carbohydrates, including combinations of different carbohydrates on one

scaffold.

The homotypic aggregation studies performed here show that certain

carbohydrate-functionalized dendrimers do manipulate cancer cell aggregation. Lower

generations of lactose functionalized dendrimers 1 and 2 inhibit HT-1080 aggregation

significantly better than competitive inhibitor lactose. LacNAc functionalized fourth

generation 8 dendrimer greatly enhances homotypic aggregation.

These homotypic aggregation studies are the initial stages of research with

carbohydrate-functionalized dendrimers and their effects on galectin influenced cancer

cells. Supplemental LacNAc studies with additional generations of dendrimer will be

performed to augment the results of this study. Additional cancer cell lines will be tested

to furthur the investigation. Also, fluorescence lifetime waveform techniques are

proposed for characterizing the glycodendrimer/galectin-3 aggregates to better

understand how the glycodendrimer/galectin clusters manipulate tumor cell aggregation.

46

CHAPTER 6

MATERIALS AND METHODS

Galectin-3 Purification

YT stock was made with 16 g tryptone peptone, 10 g yeast extract and 5 g of

NaCl dissolved in 900 mL of millipore water and set to a pH of 7.0 with 1 N NaOH. The

volume was brought up to 1 L and the solution was autoclaved. 10 mg/mL ampicillin

(amp) was aseptically added to an 80 mL portion of the YT. An overnight culture was

started from a frozen stock culture generously donated to by in 80 mL YTA (yeast,

tryptone and 10 mg/mL amp) at 37 °C and 250 rpm. The remaining 920 mL was

aseptically divided into four 1 L flasks, warmed when ready to use and 10 mg/mL of amp

was aseptically added to each. Each flask was inoculated with 20 mL of the overnight

growth. These incubated at 37 °C and 250 rpm until the optical density (O.D.) was 0.5-

2.0 at λ = 600 nm (1-2 hours). Growth was induced was a final concentration of 0.1 mM

IPTG and allowed to grow 4-5 more hours at 37 °C and 250 rpm. The growth was then

centrifuged at 2,500 rpm for 15 minutes to pellet the cells. The supernatant was poured

off. The pellet was then either resuspended with 12.5 mL of 1 X cold PBS or stored at

-70 °C. The suspension was microfluidized then centrifuged at 9,000 rpm for 10 minutes.

The supernatant was transferred to 15 mL falcon tubes and 0.15 mL of 50% glutathione

sepharose 4B slurry was added to each tube. These were incubated for 1 hour at 4 °C with

gentle agitation and then centrifuged at 500 g for 5 minutes to pellet the sepharose. The

supernatant was removed and 1 mL of 1X cold PBS added to each tube. This was then

centrifuged at 500 g for 5 minutes and the supernatant again removed. This wash was

47

repeated once more and the contents were transferred to smaller eppendorf tubes. The

contents were then washed with 1mL of cleavage buffer. A 20 µL:480 µL mixture of

Prescission Protease to cleavage buffer respectively was made. This was added to the

eppendorf tubes so that there was a total of 500 µL PP:CB mixture in 5 mL of the

Cleavage Buffer wash. The mixture incubated overnight in a rotator at 4 °C. This was

then centrifuged at 500 g for 5 minutes and the eluate collected. Each tube was washed

three times with 1 mL cold cleavage buffer and saved in -70°C.

Carbohydrate Functionalized Dendrimers

Functionalized dendrimers 3-7 were synthesized in the Cloninger lab by Dr. Mark

Wolfenden (Wolfenden Doctoral Thesis). Functionalized dendrimer 1 was synthesized by

Anna Michel (unpublished). Additional 3 was synthesized by Shannon Nissen

(unpublished). Glycodendrimer 8 was synthesized by Michael Capp (unpublished).

Cell cultures

HT-1080 human fibrosarcoma cells (ATCC) were grown in complete DMEM

supplemented with 5% heat inactivated fetal bovine serum. Cells were subcultured and

harvested with 2 mM EDTA.

Homotypic cellular aggregation assay

Duplicate samples were prepared with 0-20 μL of a 2 mg/mL stock solution of

glycosystem (lactose and 1-8) added to an eppendorf tube. PBS (phosphate buffered

solution) was then added to supplement the volume so that each sample had a

48

standardized volume of 20 μL of glycosystem/PBS. A constant amount of galectin-3 such

that the final concentration of galectin-3 per sample was 120 µg/mL was combined with

the glycodendrimer/PBS in the eppendorf tube. Confluent HT-1080 human fibrosarcoma

cells from ATCC grown in complete DMEM and were harvested using 2 mM EDTA

(ethylenediaminetetraacetic acid). Cells were then resuspended in serum free media

(SFM) so that 30 μL of SFM had 240,000 cells. 30 μL (240,000 cells) were added per

sample and to make total of 50 µL of solution present per tube. This mixture was

vortexed to mix the compounds. After 5 minutes of incubation at 37 °C on a rotator,

videos were taken of the cells directly from the eppendorf tubes at 10x magnification on

an inverted microscope. Before each video was taken the eppendorf was vortexed for five

seconds. Videos were also taken after one hour of incubation. 24, 44

Single cells were counted and the following equation was used to calculate the

extent of aggregation: 1-(Nt/Nc) ×100, where Nt represents the number of single cells in

the test suspension which is in the presence of dendrimer and Nc represents the number

of single cells in the control which is in the absence of dendrimer.24

49

REFERENCES CITED

_________________________

1. Lis, H.; Sharon, N., Lectins: Carbohydrate-specific proteins that mediate cellular

recognition. Chemical Reviews 1998, 98 (2), 637-674.

2. Klyosov, A.; Witczak, Z.; Platt, D., Galectins. Wiley: Hoboken, NJ, 2008.

3. Barondes, S. H.; Cooper, D. N. W.; Gitt, M. A.; Leffler, H., Galectins - structure

and function of a large family of animal lectins. Journal of Biological Chemistry 1994,

269 (33), 20807-20810.

4. Leffler, H.; Carlsson, S.; Hedlund, M.; Qian, Y.; Poirier, F., Introduction to

galectins. Glycoconjugate Journal 2004, 19 (7-9), 433-440.

5. Ahmad, N.; Gabius, H. J.; Andre, S.; Kaltner, H.; Sabesan, S.; Roy, R.; Liu, B. C.;

Macaluso, F.; Brewer, C. F., Galectin-3 precipitates as a pentamer with synthetic

multivalent carbohydrates and forms heterogeneous cross-linked complexes. Journal of

Biological Chemistry 2004, 279 (12), 10841-10847.

6. Nieminen, J.; Kuno, A.; Hirabayashi, J.; Sato, S., Visualization of galectin-3

oligomerization on the surface of neutrophils and endothelial cells using fluorescence

resonance energy transfer. Journal of Biological Chemistry 2007, 282, 1374-1383.

7. Mehul, B.; Bawumia, S.; Martin, S. R.; Hughes, R. C., Structure of baby hamster-

kidney carbohydrate-binding protein cbp30, an s-type animal lectin. Journal of Biological

Chemistry 1994, 269 (27), 18250-18258.

8. Hsu, D. K.; Zuberi, R. I.; Liu, F. T., Biochemical and biophysical characterization

of human recombinant ige-binding protein, an s-type animal lectin. Journal of Biological

Chemistry 1992, 267 (20), 14167-14174.

9. Ochieng, J.; Furtak, V.; Lukyanov, P., Extracellular functions of galectin-3.

Glycoconjugate Journal 2004, 19 (7-9), 527-535.

10. Song, X. Z.; Xia, B. Y.; Stowell, S. R.; Lasanajak, Y.; Smith, D. F.; Cummings,

R. D., Novel Fluorescent Glycan Microarray Strategy Reveals Ligands for Galectins.

Chemistry & Biology 2009, 16 (1), 36-47.

11. Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Hart, G. W.; E. Etzler,

M., Essentials of Glycobiology, Second Edition. Cold Spring Harbor Labratory Press:

Cold Spring Harbor, NY, 2009.

50

________________________________________________________________________

12. Yu, L. G.; Andrews, N.; Zhao, Q.; McKean, D.; Williams, J. F.; Connor, L. J.;

Gerasimenko, O. V.; Hilkens, J.; Hirabayashi, J.; Kasai, K.; Rhodes, J. M., Galectin-3

interaction with Thomsen-Friedenreich disaccharide on cancer-associated MUC1 causes

increased cancer cell endothelial adhesion. Journal of Biological Chemistry 2007, 282

(1), 773-781.

13. Takenaka, Y.; Fukumori, T.; Raz, A., Galectin-3 and metastasis. Glycoconjugate

Journal 2004, 19 (7-9), 543-549.

14. Saussez, S.; Glinoer, D.; Chantrain, G.; Pattou, F.; Carnaille, B.; Andre, S.;

Gabius, H. J.; Laurent, G., Serum galectin-1 and galectin-3 levels in benign and

malignant nodular thyroid disease. Thyroid 2008, 18 (7), 705-712.

15. Saussez, S.; Camby, I.; Toubeau, G.; Kiss, R., Galectins as modulators of tumor

progression in head and neck squamous cell carcinomas. Head and Neck-Journal for the

Sciences and Specialties of the Head and Neck 2007, 29 (9), 874-884.

16. Savin, S.; Cvejic, D.; Isic, T.; Paunovic, I.; Tatic, S.; Havelka, M., Thyroid

peroxidase and gatectin-3 immunostaining in differentiated thyroid carcinoma with

clinicopathologic correlation. Human Pathology 2008, 39 (11), 1656-1663.

17. Irimura, T.; Matsushita, Y.; Sutton, R. C.; Carralero, D.; Ohannesian, D. W.;

Cleary, K. R.; Ota, D. M.; Nicolson, G. L.; Lotan, R., Increased content of an endogenous

lactose-binding lectin in human colorectal-carcinoma progressed to metastatic stages.

Cancer Research 1991, 51 (1), 387-393.

18. Iacovazzi, P. A.; Notarnicola, M.; Caruso, M. G.; Guerra, V.; Frisullo, S.;

Altomare, D. F.; Correale, M., Serum levels of galectin-3 and its ligand 90k/mac-2bp in

colorectal cancer patients. Immunopharmacology and Immunotoxicology 2010, 32 (1),

160-164.

19. Hill, M.; Mazal, D.; Andrea Biron, V.; Pereira, L.; Ubillos, L.; Berriel, E.;

Ahmed, H.; Freire, T.; Rondan, M.; Vasta, G. R.; Liu, F.-T.; Mercedes Iglesias, M.;

Osinaga, E., A Novel Clinically Relevant Animal Model for Studying Galectin-3 and Its

Ligands During Colon Carcinogenesis. Journal of Histochemistry & Cytochemistry 2010,

58 (6), 553-565.

20. Lotan, R.; Ito, H.; Yasui, W.; Yokozaki, H.; Lotan, D.; Tahara, E., Expression of

a 31-kda lactoside-binding lectin in normal human gastric-mucosa and in primary and

metastatic gastric carcinomas. International Journal of Cancer 1994, 56 (4), 474-480.

51

________________________________________________________________________

21. Kim, S. J.; Choi, I. J.; Cheong, T. C.; Lee, S. J.; Lotan, R.; Park, S. H.; Chun, K.

H., Galectin-3 Increases Gastric Cancer Cell Motility by Up-regulating Fascin-1

Expression. Gastroenterology 2010, 138 (3), 1035-U308.

22. Saussez, S.; Decaestecker, C.; Mahillon, V.; Cludts, S.; Capouillez, A.; Chevalier,

D.; Kaltner, H.; Andre, S.; Toubeau, G.; Leroy, X.; Gabius, H. J., Galectin-3

Upregulation During Tumor Progression in Head and Neck Cancer. Laryngoscope 2008,

118 (9), 1583-1590.

23. Bradley, R., Genes and cancer course. Metastasis lecture 16. Montana State

University. Bozeman, MT. 59717, 2008.

24. Iurisci, I.; Cumashi, A.; Sherman, A. A.; Tsvetkov, Y. E.; Tinari, N.; Piccolo, E.;

D'Egidio, M.; Adamo, V.; Natoli, C.; Rabinovich, G. A.; Iacobelli, S.; Nifantiev, N. E.;

Behalf, C. I., Synthetic Inhibitors of Galectin-1 and-3 Selectively Modulate Homotypic

Cell Aggregation and Tumor Cell Apoptosis. Anticancer Research 2009, 29 (1), 403-410.

25. Matarrese, P.; Fusco, O.; Tinari, N.; Natoli, C.; Liu, F. T.; Semeraro, M. L.;

Malorni, W.; Iacobelli, S., Galectin-3 overexpression protects from apoptosis by

improving cell adhesion properties. International Journal of Cancer 2000, 85 (4), 545-

554.

26. Khaldoyanidi, S. K.; Glinsky, V. V.; Sikora, L.; Glinskii, A. B.; Mossine, V. V.;

Quinn, T. P.; Glinsky, G. V.; Sriramarao, P., MDA-MB-435 human breast carcinoma cell

homo- and heterotypic adhesion under flow conditions is mediated in part by Thomsen-

Friedenreich antigen-galectin-3 interactions. Journal of Biological Chemistry 2003, 278

(6), 4127-4134.

27. Inohara, H.; Akahani, S.; Koths, K.; Raz, A., Interactions between galectin-3 and

Mac-2-binding protein mediate cell-cell adhesion. Cancer Research 1996, 56 (19), 4530-

4534.

28. Inohara, H.; Raz, A., Functional evidence that cell-surface galectin-3 mediates

homotypic cell-adhesion. Cancer Research 1995, 55 (15), 3267-3271.

29. Nangia-Makker, P.; Honjo, Y.; Sarvis, R.; Akahani, S.; Hogan, V.; Pienta, K. J.;

Raz, A., Galectin-3 induces endothetial cell morphogenesis and angiogenesis. American

Journal of Pathology 2000, 156 (3), 899-909.

30. University, E. Hallmark of Cancer: Metastasis.

http://www.cancerquest.org/index.cfm?page=6468.

31. Warfield, P. R.; Makker, P. N.; Raz, A.; Ochieng, J., Adhesion of human breast

carcinoma to extracellular matrix proteins is modulated by galectin-3. Invasion &

Metastasis 1997, 17 (2), 101-112.

http://www.cancerquest.org/index.cfm?page=6468

52

________________________________________________________________________

32. Platt, D.; Raz, A., Modulation of the lung colonization of b16-f1 melanoma-cells

by citrus pectin. Journal of the National Cancer Institute 1992, 84 (6), 438-442.

33. Nangia-Makker, P.; Hogan, V.; Honjo, Y.; Baccarini, S.; Tait, L.; Bresalier, R.;

Raz, A., Inhibition of human cancer cell growth and metastasis in nude mice by oral

intake of modified citrus pectin. Journal of the National Cancer Institute 2002, 94 (24),

1854-1862.

34. Pienta, K. J.; Naik, H.; Akhtar, A.; Yamazaki, K.; Replogle, T. S.; Lehr, J.; Donat,

T. L.; Tait, L.; Hogan, V.; Raz, A., Inhibition of spontaneous metastasis in a rat prostate-

cancer model by oral-administration of modified citrus pectin. Journal of the National

Cancer Institute 1995, 87 (5), 348-353.

35. Inohara, H.; Raz, A., Effects of natural complex carbohydrate (citrus pectin) on

murine melanoma cell properties related to galectin-3 functions. Glycoconjugate Journal

1994, 11 (6), 527-532.

36. Hayashi, A.; Gillen, A. C.; Lott, J. R., Effects of daily oral administration of

quercetin chalcone and modified citrus pectin on implanted colon-25 tumor growth in

Balb-c mice. Altern Med Rev 2000, 5 (6), 546-52.

37. Hsieh, T. C.; Wu, J. M., Changes in cell growth, cyclin/kinase, endogenous

phosphoproteins and nm 23 gene expression in human prostatic JCA-1 cells treated with

modified citrus pectin. Biochemistry and Molecular Biology International 1995, 37 (5),

833-841.

38. Avivi-Green, C.; Madar, Z.; Schwartz, B., Pectin-enriched diet affects distribution

and expression of apoptosis-cascade proteins in colonic crypts of dimethylhydrazine-

treated rats. International Journal of Molecular Medicine 2000, 6 (6), 689-698.

39. Avivi-Green, C.; Polak-Charcon, S.; Madar, Z.; Schwartz, B., Apoptosis cascade

proteins are regulated in vivo by high intracolonic butyrate concentration: Correlation

with colon cancer inhibition. Oncology Research 2000, 12 (2), 83-95.

40. Avivi-Green, C.; Polak-Charcon, S.; Madar, Z.; Schwartz, B., Dietary regulation

and localization of apoptosis cascade proteins in the colonic crypt. Journal of Cellular

Biochemistry 2000, 77 (1), 18-29.

41. Gibson, G. R.; Roberfroid, M. B., Dietary modulation of the human colonic

microbiota - introducing the concept of prebiotics. Journal of Nutrition 1995, 125 (6),

1401-1412.

53

________________________________________________________________________

42. Vrasidas, I.; Andre, S.; Valentini, P.; Bock, C.; Lensch, M.; Kaltner, H.; Liskamp,

R. M. J.; Gabius, H. J.; Pieters, R. J., Rigidified multivalent lactose molecules and their

interactions with mammalian galectins; a route to selective inhibitors. Organic &

Biomolecular Chemistry 2003, 1 (5), 803-810.

43. Glinsky, V. V.; Glinsky, G. V.; Rittenhouse-Olson, K.; Huflejt, M. E.; Glinskii,

O. V.; Deutscher, S. L.; Quinn, T. P., The role of Thomsen-Friedenreich antigen in

adhesion of human breast and prostate cancer cells to the endothelium. Cancer Research

2001, 61 (12), 4851-4857.

44. Svarovsky, S. A.; Szekely, Z.; Barchi, J. J., Synthesis of gold nanoparticles

bearing the Thomsen-Friedenreich disaccharide: a new multivalent presentation of an

important tumor antigen. Tetrahedron-Asymmetry 2005, 16 (2), 587-598.

45. Nelson, A.; Belitsky, J. M.; Vidal, S.; Joiner, C. S.; Baum, L. G.; Stoddart, J. F.,

A self-assembled multivalent pseudopolyrotaxane for binding galectin-1. Journal of the

American Chemical Society 2004, 126 (38), 11914-11922.

46. Newkome, G. R.; Moorefield, C. N.; Vogtle, F., Dendrimers and Dendrons:

Concepts, Syntheses, Applications. Wiley-VCH: 2001.

47. Dendritech, I. www.dendritech.com

48. Wolfenden, M. L.; Cloninger, M. J., Carbohydrate-functionalized dendrimers to

investigate the predictable tunability of multivalent interactions. Bioconjugate Chemistry

2006, 17 (4), 958-966.

49. Wolfenden, M. L.; Cloninger, M. J., Study of Galectin-3 utilizing Carbohydrate-

Functionalized Dendrimers. Montana State University: Bozeman, MT, 2009.

50. Amarpreet, S.; Dhiman, A. S., Cell-Based Assay Technologies - The Road to

Success? Frost & Sullivan: Oct. 22, 2004.

51. Zou, J.; Glinsky, V. V.; Landon, L. A.; Matthews, L.; Deutscher, S. L., Peptides

specific to the galectin-3 carbohydrate recognition domain inhibit metastasis-associated

cancer cell adhesion. Carcinogenesis 2005, 26 (2), 309-318.

http://www.dendritech.com/

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