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
Julie Jeannine Sprenger
2010
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Julie Jeannine Sprenger
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.
Julie Jeannine Sprenger
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
4.7 The effects of increasing concentrations functionalized dendrimer 1 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 31
4.8 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 1.. ................................................................................................ 31
4.9 The effects of increasing concentrations functionalized dendrimer 1 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 32
4.10 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 1.. ................................................................................................ 32
4.11 The effects of increasing concentrations functionalized dendrimer 1 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 33
4.12 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 1.. ................................................................................................ 33
4.13 The effects of increasing concentrations functionalized dendrimer 2 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 34
4.14 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 2.. ................................................................................................ 34
4.15 The effects of increasing concentrations functionalized dendrimer 2 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 35
x
LIST OF FIGURES - CONTINUED
Figure
Page
4.16 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 2.. ............................................................................................... 35
4.17 The effects of increasing concentrations functionalized dendrimer 2 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 36
4.18 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 2.. ................................................................................................ 36
4.19 The effects of increasing concentrations functionalized dendrimer 4 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 37
4.20 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 4.. ................................................................................................ 37
4.21 The effects of increasing concentrations functionalized dendrimer 5 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 38
4.22 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
dendrimer 5.. ................................................................................................ 38
4.23 The effects of increasing concentrations functionalized dendrimer 8 on
galectin-3 induced homotypic HT-1080 aggregation.. ................................. 39
4.24 Pictures of HT-1080 cellular aggregation with galectin-3 and functionalized
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 monoand oligosaccharides. Lectins are capable of inducing cellular agglutination by crosslinking 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
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
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. Galectin3 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 galectin3 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, MDAMB-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 galectin3/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 MCPtreated mice was significantly reduced compared to the control group (Figure 3.1).33 The
multivalent carbohydrate, MCP, competitively blocks the galectin-3 carbohydratebinding 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) tumorassociated 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)
(A) CH2=CHCO2Me
H2NCH2CH2NH2
(A,B)
(A,B)
(B) H2NCH2CH2NH2
NH2
NH2
HN O
H
H2N N
NH2 O
H2N
H
N
O
O
O
HN
N
N
N
N
H
H
N
O
O
N
H
HN O
NH2
N
H
NH2
O NH
NH2
N
O
NH HN
N
O
N
O
HN O
HN
H2N
N
O
O
O
NH
O NH
O
H
N
N
O
N
HN O
O
NH2
NH HN
N
N
H
H2N
H NH2
N
NH2
N
NH HN
O
N
O
H2N
N
HN O
O
O NH
O
N
N
H
NH2
O NH
NH2
G(0) = blue
G(1) = red
G(2) = purple
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
0
1
2
3
4
5
6
Molecular
Measured
Weight
Diameter (Å)
(Daltons)
517
1,430
3,256
6,909
14,215
28,826
58,048
15
22
29
36
45
54
67
Surface
Groups
4
8
16
32
64
128
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 galactosidebinding 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: HT1080 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
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
16
G(2)
PAMAM
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
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
16
G(2)
PAMAM
1
Figure 4.7. The effects of increasing concentrations of functionalized dendrimer 1 on galectin-3 induced
homotypic HT-1080 aggregation. There is a significant decrease in HT-1080 aggregation at 51.2 µM of 1.
Figure 4.8. 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 8.5
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
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
16
G(2)
PAMAM
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.
Figure 4.10. 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 8.5
µM 1. D) HT-1080 cells with galectin-3 and 34.1 µM 1. E) HT-1080 cells with galectin-3 and 42.6 µM 1.
F) HT-1080 cells with galectin-3 and 51.2 µM 1.
33
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
16
G(2)
PAMAM
1
Figure 4.11. The effects of increasing concentrations of functionalized dendrimer 1 on galectin-3 induced
homotypic HT-1080 aggregation. There is a decrease in HT-1080 aggregation at 51.2 µM of 1.
Figure 4.12. 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 25.6
µM 1. D) HT-1080 cells with galectin-3 and 51.2 µM 1.
34
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
27
G(3)
PAMAM
2
Figure 4.13. The effects of increasing concentrations of functionalized dendrimer 2 on galectin-3 induced
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
cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 10.7
µM 2. D) HT-1080 cells with galectin-3 and 26.7 µM 2. E) HT-1080 cells with galectin-3 and 32.0 µM 2.
35
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
27
G(3)
PAMAM
2
Figure 4.15. The effects of increasing concentrations of functionalized dendrimer 2 on galectin-3 induced
homotypic HT-1080 aggregation. There is a decrease in HT-1080 aggregation at 26.7 µM of 2.
Figure 4.16. Pictures of HT-1080 cellular aggregation with galectin-3 and 2. A) Negative control: HT-1080
cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 10.7
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
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
27
G(3)
PAMAM
2
Figure 4.17. The effect of 32.0 µM of functionalized dendrimer 2 on galectin-3 induced homotypic HT1080 aggregation. There is a significant decrease in HT-1080 aggregation.
Figure 4.18. Pictures of HT-1080 cellular aggregation with galectin-3 and 2. A) Negative control: HT-1080
cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 32.0
µM 2.
37
OH
OH
OH
O
HO
OH
O
HO
O
O
OH
O
H
N
H
N
S
G(6)
PAMAM
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.
Figure 4.20. Pictures of HT-1080 cellular aggregation with galectin-3 and 4. A) Negative control: HT-1080
cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 3.2
µM 4. D) HT-1080 cells with galectin-3 and 6.4 µM 4.
38
HO
OH
O
O
HO
OH
O
H
N
H
N
S
G(3)
PAMAM
26
5
Figure 4.21. The effects of increasing concentrations of functionalized dendrimer 5 on galectin-3 induced
homotypic HT-1080 aggregation. There is a no significant change in HT-1080 aggregation.
Figure 4.22. Pictures of HT-1080 cellular aggregation with galectin-3 and 5. A) Negative control: HT-1080
cells alone. B) Positive control: HT-1080 cells with galectin-3. C) HT-1080 cells with galectin-3 and 5.5
µM 5. D) HT-1080 cells with galectin-3 and 15.8 µM 5. E) HT-1080 cells with galectin-3 and 31.6 µM 5.
39
OH
OH
OH
O
HO
OH
O
HO
O
O
NHAc
O
H
N
H
N
S
G(4)
PAMAM
27
8
Figure 4.23. The effects of increasing concentrations of functionalized dendrimer 8 on galectin-3 induced
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)
Lactose (1)
1(16)
1 (16)
1 (16)
2 (23)
2 (23)
2,337
8.5
42.6
51.2
26.7
32.0
2,337
136
682
819
613
736
51.2
51.2
51.2
32.0
819
819
819
736
x fold
enhancement per
compound of ≥
50% inhibition
compared to
lactose
275
55
46
88
73
x fold
enhancement
per sugar of ≥
50% inhibition
compared to
lactose
17
3.4
2.8
3.8
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 galectin3 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 functionalizeddendrimers 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 galectin3 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 0.52.0 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
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