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Effect of vicrostatin (an integrin based therapy) on canine osteosarcoma
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Effect of vicrostatin (an integrin based therapy) on canine osteosarcoma
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1
Effect of Vicrostatin (an integrin based therapy) on
canine osteosarcoma
By
Kruttika Dabke
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
August 2016
Copyright 2016 Kruttika Dabke
2
Dedication
To my family, Apoorva, and Mohit who made me who I am today.
3
Acknowledgements
I would like to express my gratitude to my advisor Dr. Markland for giving me an
opportunity to carry out research in the laboratory and also for providing me constant
guidance and support. I want to thank Dr. Clay Wang and Dr. J. Andrew MacKay for
serving as members of my thesis committee.
I would like to thank my mentor Dr. Swenson, for teaching me experimental skills
and for always being there to answer all my questions however silly they might be. His
encouragement, enthusiasm and faith in me were extremely helpful. His brilliant sense of
humor made learning a fun task. I am grateful for the help and support of my lab members
Laura, Mirna, Megan and Hershi. I would like to thank Dr. Radu for his support as well as
the amazing conversations we had which made every day in the lab more enjoyable. I
would also like to thank Dr. Clay Wang for always supporting and guiding me in the right
direction.
I owe all my accomplishments and success in my life to my parents who have truly
put my needs before their own and have always been there to support me and encourage
me to do my best. I would like to thank all my friends, especially Apoorva Oak and Mohit
Dave for their endless support and faith in my abilities, their presence made this journey
all the more enjoyable.
4
Table of Contents
Acknowledgements ........................................................................................................... 3
Table of Contents ............................................................................................................... 4
List of Tables ...................................................................................................................... 5
List of Figures ..................................................................................................................... 6
Abstract ............................................................................................................................... 6
Chapter 1: Introduction ..................................................................................................... 9
1.1 Cancer; Canine Osteosarcoma (OSA) ................................................................................ 9
1.2 Metastasis ............................................................................................................................. 10
1.3 Integrins ................................................................................................................................. 11
1.3.a Integrins- Function ................................................................................................................ 11
1.3.b Integrins- Structure ............................................................................................................... 13
1.4 Focal Adhesion Kinase (FAK) ............................................................................................ 15
1.5 Disintegrins ........................................................................................................................... 16
1.6 Vicrostatin (VCN) ................................................................................................................. 18
Chapter 2: Materials and Methods .................................................................................. 20
2.1 Cell Culture and antibodies ................................................................................................ 20
2.2 Cell Lysate ............................................................................................................................. 20
2.3 Immunoblot Analysis .......................................................................................................... 21
2.4 Phosphorylated Focal Adhesion Kinase (FAK) assay ................................................... 21
2.5 Cell migration assay ............................................................................................................ 22
2.6 Cell Adhesion assay ............................................................................................................ 23
2.7 H&E and Immunohistochemical staining ......................................................................... 24
2.8 Animals .................................................................................................................................. 24
Chapter 3: Results ............................................................................................................ 25
3.1 Detection of integrins (b3) on PARKS cell ....................................................................... 25
3.2 FAK Phosphorylation studies ............................................................................................ 26
3.3 Inhibition of cell adhesion .................................................................................................. 27
3.4 Inhibition of cell migration ................................................................................................. 29
3.5 H&E and Immunohistochemical staining ......................................................................... 31
Discussion .......................................................................................................................... 33
References: ........................................................................................................................ 37
5
List of Tables
Table1: Cell migration and adhesion assay, each box represents a well in
a 24 well plate…………………………………………………………………………23
6
List of Figures
Figure 1.1: Role of integrins in cell adhesion………………………………………………11
Figure 1.2: Different types of integrins expressed on multiple cell types…………….....13
Figure 1.3: Integrin Structure………………………………………………………………...15
Figure 1.4: Regulation of Intracellular signaling by integrins……………………………..16
Figure 1.5: Sequences of Contortrostatin, recombinant Contortrostatin, Vicrostatin and
echistatin………………………………………………………………………….19
Figure 3.1: Detection of b3 integrin and actin……………………………………………...25
Figure 3.2: Effect of VCN on Phosphorylation status of Focal Adhesion Kinase (FAK) in
PARKS cells……………………………………………………………………...26
Figure 3.3: Effect of VCN on cell adhesion, spread of PARKS cells after t=0 min…….27
Figure 3.4: Effect of VCN on cell adhesion, spread of PARKS cells after t=10 min…...28
Figure 3.5: Effect of VCN on cell adhesion, spread of PARKS cells after t=30 min…...28
Figure 3.6: Effect of VCN on cell migration of PARK cells grown on Fibronectin and
Collagen after 4 hours of incubation…………………………………………..29
Figure 3.7: Effect of VCN on cell migration of PARK cells grown on Collagen and
fibronectin after 24 hours of incubation……………………………………….30
Figure 3.8: Tissue sections probed for presence of integrin b3 and b5………………...31
Figure 3.9: H&E staining of canine osteosarcoma tissue section……………………….32
7
Abstract
Osteosarcoma (OSA) is a primary bone neoplasm which is of mesenchymal origin.
Canine OSA closely resembles human OSA in its pathogenesis, biological behavior and
histological appearance. These qualities make it an excellent candidate to model tumor
in vivo biological and preclinical studies.
A malignant tumor is characterized by its tendency to intermix with cells of various
compartments and cross tissue boundaries. This requires specific interaction with ECM
which is facilitated through integrins. Integrins physically tether cells to matrix and send
and receive molecular signals that regulate cell invasion. Integrins are overexpressed in
cancer cells and they aid in cell migration. Disintegrins represent a new class of low
molecular weight peptides which disrupt integrin function by directly binding to them.
VCN, a recombinantly produced disintegrin was developed successfully in the Markland
laboratory and tested against various cancer types including breast, ovarian and prostate
cancer.
In this report we characterize the PARKS canine osteosarcoma cell line to determine its
compatibility to disintegrin treatment. We show that VCN inhibits cell adhesion and
migration of the canine osteosarcoma cells in a dose dependent manner. FAK is a
tyrosine kinase that regulates a plethora of signaling cascades which ultimately influence
various cellular activities like migration, adhesion, proliferation and survival. Through
western blots and immunoprecipitation, we show that VCN associates with focal adhesion
kinases and increases its phosphorylation at Tyr- 397 in a dose dependent manner. In
vitro studies on VCN suggest that it has an inhibitory effect on integrins present in canine
osteosarcoma cell lines. We are trying to develop a xenograft mouse model for canine
8
osteosarcoma, for which currently there are very few established models. Higher
incidences and rapid progression of tumor malignancies leads to rapid accrual of data in
canine cancer models. These features make canines an attractive model for studying
human cancers.
9
Chapter 1: Introduction
1.1 Cancer; Canine Osteosarcoma (OSA)
Cancer is a disease defined by uncontrolled proliferation of cells. Self-sufficiency in
growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative
potential, sustained angiogenesis and tumor invasion and metastasis are the six
hallmarks of cancer (Hanahan and Weinberg 2000). Osteosarcoma (OSA), specifically,
is a high-grade primary bone neoplasm of mesenchymal origin. The incidence of OSA in
humans has been reported to be 1-3 cases per million annually, worldwide (Fuchs and
Kaser-Hotz 2007). OSA also accounts for the majority of primary bone tumors in dogs.
Canine Osteosarcoma shares biological and clinical similarities with osteosarcoma in
humans (Wycislo and Fan 2015). Canine OSA closely resembles human OSA in its
histological appearance, biological behavior, pathogenesis due to which it can become
an excellent model for application of tumor biological and preclinical studies in vivo
(Barroga et. al. 1998).
Histologically, OSA is composed of malignant mesenchymal cells of osteoblast or stem
cell lineage which produce osteoid (the un-mineralized organic component of bone).
Osteoblastic, fibroblastic, chondroblastic and telangiectatic phenotypes exist for different
histologic sub-types of OSA depending on the morphology and differentiation
characteristics of tumor cells. Classically, OSA is considered as a disease of giant breed
dogs and has a tendency to arise from the appendicular skeleton. Most likely affected
population are the middle-aged to older dogs. Biological behavior of OSA is aggressive,
with distant organ involvement as a result of metastatic disease progression. Although a
10
small population of dogs and people present with detectable lung metastasis, the eventual
development of distant metastatic foci in the absence of chemotherapy is 80% within 2
years for people and 90% within 1 year for dogs (Wycislo and Fan 2015).
A combination of chemotherapy and surgery is used as standard of care which is defined
as treatment that results in the longest median survival time for OSA (Kaser- Hotz et. al.,
2007). Although improvements in diagnostic and imaging techniques along with
chemotherapy and surgical procedures have improved outcomes in both human and
canine patients, there is still a need for effective treatment of OSA, mainly to control
metastatic disease (Morello et. al. 2011).
1.2 Metastasis
A malignant tumor is characterized by its tendency to cross tissue boundaries and
intermix with cells of various compartments. Tumor invasion and metastasis formation
require specific interactions of cells with extracellular matrix (ECM) (Stetler- Stevenson
et. al. 2000). Cancer cells develop altered affinity and avidity for ECM. The phenotypic
change is initially mediated by alterations in expression of integrins (which are cell surface
molecules), release of proteases to remodel the ECM, and secretion of new ECM
molecules. These changes activate signaling cascades that regulate gene expression,
cell adhesion, survival and cytoskeletal organization. This enables cancer cells to become
more invasive, migratory and better equipped to survive in different microenvironments
(Hood and Cheresh 2002). Different types of cell movement define distinct modes of
migration ranging from single cell migration to collective cell migration. Integrins are some
of the best characterized trans-membrane receptors that mediate dynamic interactions
between ECM and actin cytoskeleton during cell migration (Huttenlocher and Horwitz,
11
2011). Cells form different types of adhesive structures to connect with the environment.
Podosomes and invadopodia are two types of actin- rich adhesions. They not only
establish cell contact to the substratum but are also involved in matrix degradation and
hence are found in invasive cell types. Invadopodia are typically found in carcinoma cells
(Linder 2007).
Figure 1.1: Schematic showing major compositional and structural differences between
focal adhesions and invadopodia and role of integrins in both types of structures (Source:
Huttenlocher and Horwitz, 2011). As seen in the Fig 1.1, linkage between integrins and
actin could occur through talin or a-actinin in focal adhesions. Podosomes and
invadopodia are entirely different classes of migratory-related adhesions. They differ
significantly in organization and composition from focal adhesions but integrins are
integral to both types of adhesions (Linder 2007).
1.3 Integrins
1.3.a Integrins- Function
Integrins are large heterodimeric cell surface receptors found in a wide range of animal
species from sponges to mammals. They are involved in fundamental cellular processes
such as survival, differentiation, proliferation, migration and attachment (xiong et. al.
12
2001). Integrins have a multitude of intracellular effects on the organization of actin-
containing cytoskeleton as well as roles in variety of signaling processes which in turn
effects a cell’s ability to proliferate, survive and differentiate. A complex series of steps
from interaction of integrins with extracellular ligands, localization of activation
cytoskeletal molecules and activation of signaling pathways leads to eventual regulation
of gene expression (Miyamoto et. al. 1995). Adhesion plays several roles in cell migration.
It links the extracellular substratum to actinomyosin filaments generating traction and
organizes signaling networks that also regulate cellular processes like proliferation, gene
expression, and cell survival (Huttenlocher and Horwitz 2011 ). Integrins not only
physically tether cells to the matrix but also send and receive molecular signals that
regulate cell invasion and migration (Hood and Cheresh 2002).
Integrins are also involved in growth and survival of newly forming blood vessels. They
are overexpressed on the endothelial cell surface in order to facilitate their growth and
survival (Weis and Cheresh 2011). During embryonic development, successful vascular
development depends on fibronectin and its major receptor avb1. It does not depend on
avb3, avb5 and a6b4 which are in fact central regulators of postnatal angiogenesis (serini
et. al. 2005). Two types of integrins are involved in disparate pathways namely avb3 and
avb5. The function of avb3 is required for angiogenesis and is induced by basic fibroblast
growth factor (bFGF) or tumor necrosis factor a (TNF-a), whereas angiogenesis induced
by vascular endothelial growth factor (VEGF) or transforming growth factor (TGF-a)
requires the function of integrin a5b5 (Weis and Cheresh 2011).
13
Figure 1.2: Integrins expressed on multiple cell types which contribute to angiogenesis
and tumor progression (source: Weis and Cheresh 2011). Newly forming endothelial cells
express a unique profile of integrins that can be used to target vascular proliferation.
Integrins influence pericyte coverage of maturing blood vessels. Tumor cells change
integrin expression profile to enhance their ability to migrate, invade metastasize and
survive in hostile environments. Fibroblasts direct synthesis of extracellular matrix
proteins and growth factors in the tumor stroma via integrin signaling. Integrin expression
profiles of normal cells are distinct to those within remodeling tissues (Weis and Cheresh
2011).
1.3.b Integrins- Structure
Integrins a diverse family of glycoproteins are ab heterodimers; each subunit crosses the
membrane once with most of the polypeptide in extracellular space and two short
domains in the cytoplasm. 8b subunits combine with 18a subunits to form 24 distinct
integrins (Hynes 2002). Each of these receptors has different binding properties and
different tissue distribution. Integrins also adopt two states characterized by low or high
14
affinity to ligands. Outside-in signaling occurs as a consequence of binding extracellular
ligands and inside-out signaling is a result of interactions with proteins bound within
cytoplasmic domains (Adair and Yeager, 2002). They have a large extracellular domain,
single membrane spanning helix and a short cytoplasmic tail. The subunits are
constructed from several domains with flexible linker domains joining them. Structural
studies of intact ectodomains postulate that an upright structure as well as a bent structure
could exist which is referred to as the ‘switchblade’ model (Campbell and Humphries,
2011). Some integrins are promiscuous in terms of binding partners, binding to several
ECM proteins. The a subunit consists of four or five extracellular domains which are; a
seven bladed b propeller, a thigh domain and two calf domains. More than half of the
integrin a chains have an a-I domain which is around 200 amino acids long (Larson et.
al. 1989). The I- domain is similar to the Von Willebrand factor type A domain. The last
three or four blades of the b-propeller domain contain Ca
2+
binding domains on the lower
side of the blades which face away from ligand-binding surface. Ligand binding has been
shown to be influenced by Ca
2+
binding to these sites (Oxvig and Springer 1998;
Humphries et. al. 2003). The structure of a-I domain suggests that integrin binding
involves a Mg
2+
ion, at a “metal-ion-dependent adhesion site” (MIDAS) (Lee et. al. 1995).
The b subunit has seven domains namely, b-I domain, hybrid domain, plexin-semaphorin-
integrin (PSI) domain, cysteine-rich epidermal growth factor (EGF) modules and a b-tail
domain. The b-I domain is similar to the a-I domain and is inserted into the hybrid domain
(Xiao et. al. 2004; Arnaout et al. 2005). As mentioned before, binding of ligands is
dependent on Mg
2+
, Ca
2+
and Mn
2+
ions.
15
a. Inactive b. Activated
Figure 1.3: Integrin structure; Cartoon representation of the bent and upright
conformations with approximate dimensions Fig 1.3-a, b represents active and inactive
structure of integrin respectively (Source: Campbell and Humphries, 2011). There has
been a general acceptance that conformations of integrin structure like Fig 1.3.a and Fig
1.3.b are possible because of observed flexibility in studies by electron microscopy (EM)
and the existence of conformationally sensitive antibody recognition sites. Structural
studies of intact ectodomains of integrins all postulated that an upright structure could
exist as well as the bent structure (Campbell and Humphries, 2011).
1.4 Focal Adhesion Kinase (FAK)
Focal Adhesion Kinase (FAK) plays an important role in transducing external signals
to the inside of the cell. FAK gets activated by integrin engagement and binds to paxillin,
vitronectin and talin within the cell. Normally, FAK proteins are auto inhibited mainly
because of intramolecular interactions between kinase and the amino terminal FREM
domain. This inhibitory action is disrupted by integrins causing transient dimerization of
FAK molecules. This causes increased phosphorylation on Tyr-397 and produces a high
16
affinity anchoring site for binding to Src. This further leads to formation of a stable FAK-
Src complex which in turn phosphorylates many other substrates like CAS, paxillin, and
p190RhoGAP. These molecules have a central role in reorganization of the actin
cytoskeleton and cell migration (Parsons 2003, Zhao and Guan 2009).
Figure 1.4: Regulation of Intracellular signaling by integrins (source: Hood and Cheresh,
2002). Integrin engagement leads to a wide variety of downstream events within a cell.
They can influence different signaling cascades which ultimately influences cell migration
and invasion. As seen in Fig 1.4, Focal adhesion kinases and Src-family Kinases are
some of the first molecules activated by integrin engagement.
Abbreviations: FAK (Focal Adhesion Kinase); PKC (Protein Kinase C); SRC (Src-family
kinase); ERK (Extra-cellular regulated kinase); MLCK (Myosin light-chain kinase); RAC
(Ras-related C3 botulinum toxin substrate); RAF (Rapidly Accelerated Fibrosarcoma);
PI3-K (Phosphotidylinositol-3-kinase).
1.5 Disintegrins
Most integrin receptors have a characteristic feature of binding to a wide variety
of ligands. Moreover, many cell surface adhesion proteins and extracellular matrix
17
proteins bind to multiple integrin receptors (Humphries et. al., 2006). Many adhesive
proteins including fibronectin, vitronectin, osteopontin, collagens and von Wille-brand
factor present in extracellular matrices and in the blood, contain a tripeptide; arginine-
glycine-aspartic acid (RGD) as their cell recognition site (Ruoslahti and Pierschbacher
1987). All aV integrins, two b1 and aIIb3 integrins have the ability to recognize ligands
which contain RGD tri-peptide at their active site (Humphries et. al., 2006).
Disintegrins represent a new class of low molecular weight, RGD-containing cysteine-rich
peptides which were isolated from venom of snakes mostly belonging to the viperidae
family. Amino acid sequence and functional studies of the different disintegrins suggests
that the RGD sequence functions as a cell recognition site. The 3-D configuration of the
proteins is determined by the appropriate pairing of the cysteine residues (Gould et. al.
1990).
Based on their anti- angiogenic and anti-metastatic effects demonstrated in various
experimental settings, these small polypeptides hold a significant translational potential
as anti-cancer agents (Minea et. al. 2010). Dr. Markland’s lab was successful in isolating
a snake venom disintegrin called contortrostatin (CN) which was shown to affect tumor
cell growth directly as well as through inhibition of angiogenesis (Trikha et. al. 1994).
Despite all the advantages, the problem with CN was its availability. Snake venom has a
very limited amount of CN making it difficult to use as a therapeutic agent. Later, Dr.
Minea in Markland’s lab designed and engineered a novel recombinant disintegrin called
vicrostatin to resolve this issue.
18
1.6 Vicrostatin (VCN)
Vicrostatin (VCN) is a chimeric disintegrin generated recombinantly by grafting the C-
terminal tail of viperid snake venom disintegrin echistatin to the sequence of crotalid
disintegrin contortrostatin. This novel disintegrin can be produced in high quantities as an
active polypeptide in origami B. E. coli. This strain has been uniquely designed to
overcome the shortcomings of disulfide-rich recombinant protein production (Minea et. al.
2010). VCN retains the integrin binding specificity of both its parental molecules, but with
a different binding affinity profile. It not only competes for the same integrin receptors that
are preferentially upregulated in tumor microenvironment, but VCN also exerts a potent
inhibitory effect on endothelial cell migration and tube formation in a dose dependent
manner. It acts by forcing the cells to undergo significant actin cytoskeleton reorganization
when exposed to this agent in vitro. VCN has also been shown to inhibit motility of breast
cancer cells in vitro (Minea et. al. 2011).
19
Figure 1.5: Comparison between contortrostatin, recombinant contortrostatin, vicrostatin
and echistatin sequences (Source: Minea et. al., 2010). As seen in Fig 1.5, VCN as a
molecule is a monomer and not a homodimer as in case of CN. The figure shows how
the C-terminal tale of VCN was generated by modification of CN and also shows the Arg-
Gly-Asp tripeptide motifs in bold with the graft in the structure of VCN underlined.
VCN has been tested on different types of human cancers namely, breast cancer,
prostate cancer, ovarian cancer and glioma, in the Markland lab. It has also been shown
to have an effective role in inhibiting the process of platelet aggregation.
The main aim of my study is to (1) characterize proteins in ‘PARKS’ a canine
osteosarcoma cell line (2) identify different types of integrins that might be present (3) test
a dose-dependent effect of VCN on ‘PARKS’ cells in vitro and (4) develop a canine OSA
murine model to test the efficacy of VCN in vivo.
20
Chapter 2: Materials and Methods
2.1 Cell Culture and antibodies
PARKS a canine osteosarcoma cell line was obtained from Dr. Colorado State
University, School of Veterinary Medicine. PARKS cells were grown in Dulbecco’s
Modified Eagle’s medium (DMEM) supplemented with 10% Fetal Bovine Serum Albumin
(FBS), 100 U/ml penicillin and 0.1mg/ml streptomycin. They were incubated in a
humidified atmosphere 37
0
C and 5% CO
2
in tissue culture flasks. The cells were grown
to 80% confluency before being passaged.
Antibodies for the assays were purchased from Santa Cruz Biotechnologies (b3:H-96;
FAK: A-17; p-FAK: Tyr 397-R; Actin: I-19). The secondary antibodies labeled with infra-
red dyes were purchased from Li-Cor Biosciences (Donkey anti-goat: 926-32214, Donkey
anti- mouse: 926-32212, Donkey anti- rabbit: 926- 32213)
2.2 Cell Lysate
PARKS cells were grown to 80% confluency in tissue culture flasks. Fresh cell lysate
was prepared using freeze-thaw method in liquid nitrogen. The cell pellets obtained from
tissue culture flasks were resuspended in 200µl of PBS in 1.5ml centrifuge tubes in the
presence of 1X protease inhibitor cocktail (Roche). The cell suspensions were frozen in
liquid nitrogen and then thawed at 37
0
C. Multiple cycles of this were carried out for
efficient lysis. Supernatant was collected after spinning the tubes for 5 minutes at 14,000
rpm at room temperature.
21
2.3 Immunoblot Analysis
Cell lysate was obtained as described previously. Protein samples were resolved by
SDS- Polyacrylamide gel electrophoresis. The gel was run at 150V for 60-90 min, proteins
were transferred to nitro-cellulose membrane using wet transfer method and blocked with
15µl blocking buffer (1% BSA in PBS containing 0.05% Tween20) for 1 hour at room
temperature. After washing the membrane was than incubated with primary antibody of
interest at a concentration of 1:1000 overnight at 4
0
C on a rotating device. Post incubation
the blots was washed in 0.2% TBST (Tris-buffered Saline and Tween 20); the membrane
was incubated with secondary antibody of interest at a concentration of 1:500. Lic-cor
Bioscience Odyssey infrared imaging system was used for detection.
2.4 Phosphorylated Focal Adhesion Kinase (FAK) assay
Cells were grown to 80% confluency in tissue culture flasks. Cells were collected using
0.05% trypsin, were resuspended in 1ml serum free media in 1.7ml centrifuge tubes and
incubated for 4 hours at 37
0
C on a rotating device. The cells were then incubated with
varying concentrations of VCN (0, 100, 500 and 1000nM) for 1 hour at 37
0
C on a rotating
device. Cell lysate was obtained by freeze-thaw method as described previously. 8 µg
FAK (A-17) antibody was added to each tube for immunoprecipitation and kept overnight
at 4
0
C on a rotating device. 20µg agarose beads were added to the tubes and kept
overnight at 4
0
C on a rotating device. The agarose beads were centrifuged at 2500g at
4
0
C and washed 3 times with 1ml PBS. Loading buffer was added to each tube and
samples were incubated at 50
0
C for 10 minutes and then placed in boiling water for 5
min. Supernatant was collected after centrifugation at 4000g at 37
0
C. Protein samples
22
were resolved by SDS- Polyacrylamide gel electrophoresis. The gel was run at 100v for
60-90 minutes, at room temperature, and proteins were transferred to nitro-cellulose
membrane using wet transfer method. The membrane was blocked with 15µl of blocking
buffer (1% BSA in PBS containing 0.05% Tween20) for 1 hour at room temperature. The
membrane was than incubated with primary antibody at a concentration of 1:1000
overnight at 4
0
C. Post incubation membrane was washed in 0.2% TBST, the membrane
was incubated with secondary antibody at a concentration of 1:500. Lic-cor Bioscience
Odyssey infrared imaging system was used for detection.
2.5 Cell migration assay
24 well plates were coated with fibronectin and collagen type-IV as shown in Table1
at a concentration of 10µg/cm
2
. Each well surface was covered with 200ul of fibronectin
or collagen and incubated for 2 hours at 37
0
C. Unbound ECM was removed and the wells
were blocked using 1% BSA in PBS and incubated for 1 hour at 37
0
C. The wells were
than washed once with PBS. Cells were grown to 80% confluency in a 24 well plate with
a seeding density of 0.07X10
6
cells/ml. A scratch was made with a 200ul pipette tip
diagonally across the well after removing the media. The wells were then filled with DMEM
(containing 10% FBS, 100 U/ml penicillin and 0.1mg/ml streptomycin) and varying
concentrations of VCN as seen in Table1. The experiment was performed in duplicate.
Images were taken of every well on phase contrast optical microscope with appropriate
restriction markers after 4 hours of incubation. Images were captured again with
restriction markers after 24 hours and compared for effect of drug.
23
Table1: Cell migration and adhesion assay, each box represents a well in a 24 well plate
2.6 Cell Adhesion assay
Cells were allowed to grow to 80% confluency in tissue culture flasks. Approximately
4X10
6
cells/ml per flask were collected using 0.05% Trypsin, pooled together and
resuspended in 1ml serum free media in 1.7ml centrifuge tubes and incubated for 4 hours
at 37
0
C on a rotating device. The cells were than incubated with varying concentrations
of VCN (0, 100 and 1000nm) for 1 hour at 37
0
C on a rotating device. The 24 well plates
were previously coated with fibronectin and collagen type-IV as shown in Table1. Each
well surface was covered with 200ul of fibronectin or collagen type-IV and incubated for
2 hours at 37
0
C. Unbound ECM was removed and the wells were blocked using 1% BSA
in PBS and incubated for one hour at 37
0
C. The wells were than washed once with PBS.
Volume of cell suspension corresponding to 0.1X10
6
cells/ml (»6-7 µl) + 400µl of media
was added to each well in a 24well plate. Cells were allowed to interact with fibronectin
and collagen type-IV for varying intervals of time (0, 10, 30 min). The media was aspirated
and each well was washed with 200µl PBS after each time interval before imaging.
Images were captured at each time interval using registration markers using a phase
contrast microscope.
Concentration of VCN in nm
Collagen
Type- IV
0nm 100nm 1000nm 0nm 100nm 1000nm
Fibronectin 0nm 100nm 1000nm 0nm 100nm 1000nm
24
2.7 H&E and Immunohistochemical staining
Frozen canine osteosarcoma tumor samples were obtained from Colorado State
University, Veterinary School of Medicine. They were embedded using Optimal Cutting
Temperature (OCT) embedding compound and were sliced using microtome. The
sections were fixed onto slides using alcohol as fixative and used for
immunohistochemistry and H&E staining. Enzo immunohistochemistry kit was used to
probe the slides with b3 and b5 antibodies.
2.8 Animals
Animals used in the study were kept in a pathogen-free environment, and fed with
sterilized food and water. All animals were maintained at the University of Southern
California (Los Angeles, CA) following National Institutes of Health guidelines on use of
laboratory animals and with an approved protocol by the University of Southern California
Institutional Animal Care and Use Committee
25
Chapter 3: Results
3.1 Detection of integrins (b3) on PARKS cell
Vicrostatin is a disintegrin which interacts with the integrins on tumors to inhibit
metastasis. Thus, it was important to determine the presence of integrins on the given
canine osteosarcoma in vitro model. Western blotting, using antibodies specific for
integrin b3, for the PARKS cell lysate, shows the presence of integrins, indicating its
compatibility for VCN treatment.
Lane I Lane 2
A
Figure 3.1: Western blot showing the presence of integrin b3 on PARK cells. The
experiment was performed in duplicate.
Western blot assay was performed using secondary antibodies labeled with infra-red dyes
and detected using Li-cor Biosciences Odyssey infrared imaging system. A band was
clearly visible on the nitro-cellulose membrane after imaging at around 100 kD and around
40 kD indicating the presence of integrin b3 and actin.
Integrin b3- 100 kD
Actin 42 kD
26
3.2 FAK Phosphorylation studies
FAK is a tyrosine kinase that regulates a plethora of signaling cascades which
ultimately influences cellular activities like migration, adhesion, proliferation and survival.
FAK is activated by integrin engagement leading to auto-phosphorylation at Tyr-397 and
causes subsequent downstream events.
In order to understand the interaction of VCN and integrins in PARKS cell line we are
assaying the phosphorylation status of FAK on VCN treatment.
1000nM 500nM 100nM 0nM
A pFAK- Y397 125 kD
B FAK- 125 kD
Figure 3.2: Effect of VCN on Phosphorylation status of Focal Adhesion Kinase (FAK) in
PARKS cells.
Panel A shows western blot image showing levels of pFAK- Y397 treated with VCN. 100
nM concentration of VCN shows an increase in phosphorylation level, however, 500 nM
and 1000 nM concentration of VCN shows significant increase in phosphorylation. Panel
B shows FAK levels after being probed with FAK monoclonal antibody. VCN engages
integrins agonistically and hence we see an increase in phosphorylation of FAK at Tyr-
397 in a dose dependent manner and there is no change in FAK protein observed in panel
B.
27
3.3 Inhibition of cell adhesion
Integrins interact with various ECM proteins to facilitate cell adhesion and
migration. It has been shown from previous studies that VCN acts as an ECM mimetic
disrupting the integrin interaction with ECM proteins. In order to understand the effect of
VCN on cell migration we performed a cell adhesion assay on PARKS cells which were
cultured on ECM proteins.
No Treatment 100 nM VCN 1000 nM VCN
Figure 3.3: Effect of VCN on cell adhesion and spread of PARKS cells after time=0 min
Fewer cells were found to be attached to the matrix after 0 min in treated wells and
comparatively more number of cells are seen to attach to the matrix in untreated well.
28
No Treatment 100 nM VCN 1000 nM VCN
Figure 3.4: Effect of VCN on cell adhesion and spread of PARKS cells after time=10 min
Many cells which were untreated were attached to the matrix at 10min, fewer cells were
attached after treatment with 100nM of VCN and still fewer cells were found attached to
the matrix after being treated with 1000nM VCN concentration at 10 min.
No Treatment 100 nM of VCN 1000 nM VCN
Figure 3.5: Effect of VCN on cell adhesion and spread of PARKS cells after time=30 min
Many more untreated cells were attached to matrix at 30 min as compared to those
attached at 10 min and less cells were seen attached to the matrix after being treated
with 100nM VCN and still fewer cells were attached after further being treated with
1000nM VCN at 30 min.
29
3.4 Inhibition of cell migration
In order to understand the effect of VCN on cell migration we performed a scratch
assay on PARKS cells which were cultured on different ECM proteins namely collagen
type IV and fibronectin.
Collagen: No Treatment 100 nM of VCN 1000 nM VCN
Fibronectin: No Treatment 100nM of VCN 1000nM of VCN
Figure 3.6: Effect of VCN on cell migration of PARK cells grown on Fibronectin and
Collagen after 4 hours of incubation
30
Collagen: No Treatment 100 nM of VCN 1000 nM VCN
Fibronectin: No Treatment 100nM of VCN 1000nM of VCN
Figure 3.7: Effect of VCN on cell migration of PARK cells grown on Collagen and
fibronectin after 24 hours of incubation
After 24 hours of incubation, more cells appear to have migrated in wells without
treatment, less number of cells migrated in wells which were treated with 100nM
concentration of VCN and significant inhibition of cell migration was seen in wells treated
with 1000nM concentration of VCN.
31
3.5 H&E and Immunohistochemical staining
Immunohistochemistry for integrin b3 and b5 and H&E staining was performed on
histological sections of canine osteosarcoma samples obtained Colorado State
University, School of Veterinary Medicine. Immunohistochemistry data indicates the
presence of integrins in the histological section. Since VCN targets integrins in cancer
cells, this finding indicates that VCN can be used in vivo as a potential therapeutic
molecule against canine osteosarcoma.
Integrin b3 Integrin b5
Figure 3.8 Immunohistochemical staining of integrin b3 and b5
Integrin b3 and b5 are found to be abundantly present in histological sections.
32
Figure 3.9 H&E staining of canine osteosarcoma tissue section. The H&E staining of
canine osteosarcoma tissue section. Hematoxylin (dark blue/ violet stain) binds to
basophilic substances like DNA/RNA. Eosin (red/ pink stain) binds to acidophilic
substances like most of the proteins in the cytoplasm.
33
Discussion
Cancer is a major health problem in large parts of the world including United States
and it has been statistically seen that 1 in 7 deaths in the world and 1 in 4 deaths in United
States is caused by cancer (Siegel et. al. 2013). Osteosarcoma is the most common
primary bone tumor found in dogs with an estimated 10,000 dogs diagnosed with OSA
every year (Homes et. al. 2015). Canine osteosarcoma and human osteosarcoma have
many features in common making the study of canine osteosarcoma doubly beneficial in
terms of developing a therapy for canine as well as human OSA.
The predominant cause of morbidity and mortality associated with different types of
cancer is the emergence of metastasis to distant organs. In many types of metastatic
cancers, it happens that by the time the primary cancer is detected and diagnosed,
metastasis takes place to distant sites making it really difficult to combat the disease
(Fidler 1985). Cell migration has been acknowledged to play a critical role in tumor
metastasis (Hanahan and Weinberg 2000). Thus, the migratory machinery used by the
cell for movement can be an attractive target and can be exploited to win the war against
cancer.
Integrins are an important class of molecules and have often been targets of interest for
treatment of cancer notably because they have the capacity to conduct inside-out and
outside-in signaling (Hynes 2002). Also integrins are an integral part of many
intramolecular interactions such as crosstalk with growth factors, cytokines and also
cooperation with oncogenes. In addition to this, integrins also play an important role in
formation of focal adhesion kinases which in turn play a role in cancer metastasis
(Trusolino et. al. 2001, Guo et. al. 2006).
34
Disintegrins are low molecular weight compounds that are recognized by integrin
molecules due to their RGD sequence, they act as integrin antagonists. Vicrostatin is a
recombinant snake venom disintegrin produced in our lab which also contains the RGD
binding domain. This report shows that VCN is involved in upregulating the
phosphorylation of focal adhesion kinase in a dose dependent manner. In this report VCN
is shown to affect cell adhesion and spreading of the ‘PARKS’ canine osteosarcoma cell
line.
To study the effect of VCN on metastasis, both cell adhesion and cell migration were
investigated. Fibronectin and collagen coated plates were employed to see the effects on
cells after being treated with VCN. Both Fibronectin and collagen are ECM matrix proteins
which were used to mimic the in vivo tumor microenvironment. To study the effect of VCN
on cell adhesion, serum starved PARKS cells kept in suspension in serum free media
and receiving no external input via integrins other than disintegrin treatment, were plated
on collagen for different durations of time. It was observed that untreated cells showed
attachment to the matrix whereas VCN treated cells showed a loss of attachment in a
dose as well as time dependent manner. This indicated that VCN disrupted cell adhesion
at higher concentration and longer durations of treatment. To study the effect on cell
migration, PARKS cells were allowed to grow to confluency in 24 well plates coated with
fibronectin or collagen. A scratch was made using a 200ul pipette tip and different
concentrations of VCN were added to the media. After 24-hour incubation it was observed
that VCN prevented the migration of PARKS cells in a dose dependent manner.
After determining the effect of VCN on cell migration and adhesion, the next logical step
was to determine the mechanism with which VCN disrupts these cellular activities. To
35
examine this, immunoprecipitation and western blot was employed to study the
intracellular binding partners of integrins and to see weather treatment of cells with VCN
has an effect on the recruitment of these binding partners.
The primary molecule we studied in the signaling pathway was Focal Adhesion
Kinase. This was because FAK plays a major role in cellular adhesion. FAK auto
phosphorylates in response to integrin engagement. Under normal circumstances the
FAK molecule is auto inhibited due to intramolecular interactions. However, integrins are
involved in disrupting this auto inhibitory interaction which causes activation of FAK and
phosphorylation at its Tyr-397 (Zhao and Guan 2009).
The data obtained in this study shows that PARKS cells exposed to no treatment showed
normal levels of phosphorylated FAK. However, PARKS cells treated with VCN at varying
concentrations i.e. 100 nM, 1000 nM, showed increase in phosphorylation of FAK. To
explain these concentration dependent increases in phosphorylation of FAK and their
interpretations, it can be hypothesized that: 1) VCN like CN (Minea et. al. 2010) acts as
an ECM mimetic, binding to integrins in a manner similar to that of ECM proteins and
causing similar signaling cascades to occur. 2) But as VCN is a ECM mimetic, it prevents
the cells from attaching to the ECM and the cells lose their ability to adhere and migrate.
After determining the effect of VCN on PARKS cells in vitro, it was important to determine
its effects in vivo. As a part of our study, we are developing a xenograft mouse model for
canine osteosarcoma, for which currently there are very few established models. We
found the presence of integrin b3 and b5 on histological sections of canine osteosarcoma
samples obtained from Colorado State University, School of Veterinary Medicine, using
immunohistochemistry. Our findings of RGD-reactive integrins in the canine
36
osteosarcoma tissue indicates that VCN can be used as a potential therapeutic molecule
against canine osteosarcoma. This data will further enable us to compare the histology
and pathology of canine osteosarcoma tissues with that of the tumors obtained from our
xenograft mouse model.
Cell adhesion molecules play a dynamic role in cancer disease progression and integrins
are one of the important players in cell adhesion and migration. Integrins are thus
becoming potential targets for therapeutics. This study indicates that VCN has an adverse
effect on canine osteosarcoma cell migration and adhesion in a dose dependent manner.
We demonstrate and evaluate the potential of VCN to be used as an anti-cancer
therapeutic agent. In addition to that, the study indicates that a xenograft mouse model
of canine osteosarcoma could aid in our understanding of human osteosarcoma. Further
studies, both in-vitro and in-vivo are underway to elucidate the therapeutic efficacy of
VCN and its clinical potential as a novel drug molecule.
Currently in vivo studies are being carried out with VCN treatment being given
intravenously to our canine osteosarcoma xenograft mouse model. The results for this
study are awaited. For future studies, vicrostatin can be used to treat the xenograft tumors
using either intra-tumor injections or liposomal VCN. This will enable us to compare
results from the different strategies used for treatment and determine the most efficacious
option for the same.
37
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Abstract (if available)
Abstract
Osteosarcoma (OSA) is a primary bone neoplasm which is of mesenchymal origin. Canine OSA closely resembles human OSA in its pathogenesis, biological behavior and histological appearance. These qualities make it an excellent candidate to model tumor in vivo biological and preclinical studies. A malignant tumor is characterized by its tendency to intermix with cells of various compartments and cross tissue boundaries. This requires specific interaction with ECM which is facilitated through integrins. Integrins physically tether cells to matrix and send and receive molecular signals that regulate cell invasion. Integrins are overexpressed in cancer cells and they aid in cell migration. Disintegrins represent a new class of low molecular weight peptides which disrupt integrin function by directly binding to them. VCN, a recombinantly produced disintegrin was developed successfully in the Markland laboratory and tested against various cancer types including breast, ovarian and prostate cancer. ❧ In this report we characterize the PARKS canine osteosarcoma cell line to determine its compatibility to disintegrin treatment. We show that VCN inhibits cell adhesion and migration of the canine osteosarcoma cells in a dose dependent manner. FAK is a tyrosine kinase that regulates a plethora of signaling cascades which ultimately influence various cellular activities like migration, adhesion, proliferation and survival. Through western blots and immunoprecipitation, we show that VCN associates with focal adhesion kinases and increases its phosphorylation at Tyr- 397 in a dose dependent manner. In vitro studies on VCN suggest that it has an inhibitory effect on integrins present in canine osteosarcoma cell lines. We are trying to develop a xenograft mouse model for canine osteosarcoma, for which currently there are very few established models. Higher incidences and rapid progression of tumor malignancies leads to rapid accrual of data in canine cancer models. These features make canines an attractive model for studying human cancers.
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Dabke, Kruttika
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Effect of vicrostatin (an integrin based therapy) on canine osteosarcoma
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Molecular Pharmacology and Toxicology
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