University of Southern California Dissertations and Theses

Quantitative evaluation of the effectiveness of an integrin antagonist, vicrostatin, in cell migration

(USC Thesis Other)

Quantitative evaluation of the effectiveness of an integrin antagonist, vicrostatin, in cell migration

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content QUANTITATIVE EVALUATION OF THE EFFECTIVENESS OF
AN INTEGRIN ANTAGONIST, VICROSTATIN, IN CELL MIGRATION

by

Chien-Yu Chen

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
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2013

Copyright 2013 Chien-Yu Chen

i
Table of Contents
Abbreviations ii
List of Figures iii
Abstract iv
Introduction 1
Materials and Methods 6
Results 13
Discussion 27
References 32

ii
Abbreviations
Bovine serum albumin (BSA)
Cytochalasin D (CytoD)
Deionized H
2
O (DI H
2
O)
Dulbecco’s modified Eagle’s Medium (DMEM)
Extracellular matrix (ECM)
Fetal bovine serum (FBS)
Intraperitoneal (IP)
Phosphate buffered saline (PBS)
Roswell park memorial institute medium (RPMI)
Vicrostatin (VCN)

iii
List of Figures
Figure 1. Examples of colloidal gold-coated surface and the migration tracks 15
Figure 2. Quantitative evaluation of MDA-MB-231, Hey and SKOV-3
cell migration 18
Figure 3. Hey and MDA-MB-231 cell migration with the effect of VCN 21
Figure 4. Inhibition of Hey cell migration by VCN 22
Figure 5. Inhibition of MDA-MB-231 cell migration by VCN 23
Figure 6. Macroscopic evaluation of tumor burden 25
Figure 7. Kaplan-Meier curve for OVCAR-3 tumor model 26

iv
Abstract
Cell migration underlies tissue formation, maintenance and regeneration as well
as pathological condition such as cancer invasion and metastasis. The integrin
family of cell adhesion proteins promotes the attachment and migration of cells
on the surrounding extracellular matrix (ECM). Through signals transduced upon
integrin ligation by ECM proteins, this family of proteins plays key roles in
regulating tumor growth, angiogenesis as well as metastasis. During the last 15
years, the Markland laboratory has isolated and recombinantly produced several
disintegrin species from southern copperhead snake venom, which act as
integrin antagonists with the ability to disrupt integrin functions through direct
binding. In this study, I examined the anti-migratory effect of Vicrostatin (VCN), a
rationally designed and sequence-engineered disintegrin, with both in vitro
quantifiable cell migration assay and in vivo human ovarian cancer xenograft
mouse model. The results show that VCN at 100 nM and above significantly
inhibits in vitro MDA-MB-231 and Hey cell migrations. In separate studies, it was
shown in the lab that 5 mg VCN in slow-release gel by weekly intraperitoneal
injection prevents in vivo tumor dissemination and increases host survival. These
findings underscore the potential of VCN in anti-metastatic and anti–tumorigenic
action as a novel treatment for breast and ovarian cancer.

1
Introduction
Cellular motility is a fundamental biological event necessary for cell
translocation during normal processes such as tissue development, wound
healing and immune surveillance. In most environments, cell movement starts
with extension of the cell membrane followed by the formation of new
adhesions at the cell front that interconnect the actin cytoskeleton to the
substratum, generation of traction forces that pull the cell forwards and
disassembly of adhesions at the cell posterior allowing for cyclic reformation of
adhesions on the leading edge of the cell (Friedl and Wolf, 2009). In normal cells
and cellular processes this action is tightly controlled and purposeful.
Deregulation of the process lead to exacerbated tumor formation and cancer
metastasis.
Cancer metastasis promotes the spread of tumors to local and distant sites
away from primary tumors. Due to spread to remote and often undetectable
sites, in the early stages, cancer metastasis is the main cause of the morbidity
and mortality in cancer patients. For this reason, controlling tumor cell migration
is a central issue in cancer treatment. Although normal and tumor cells share
many of the molecular mechanisms of cell migration, tumor cell migration is
uncontrolled, randomly oriented and not coordinated. Tumor cells migrate in
groups without clear organization or as individual cells (Sahai, 2005). Despite the
fact that cells express various cell surface adhesion receptors, including integrins,

2
syndecans, other proteoglycans, cadherins and cell adhesion molecules, to date,
the integrin family of transmembrane heterodimeric receptors is the best
studied and plays a prominent and integral role in cell migration.
Integrins are heterodimeric glycoproteins spanning across plasma
membrane, composed of noncovalently associated α and β subunits. Eight
different β and eighteen different α subunits have been described, forming an
array of twenty-four different integrin heterodimers, each of which supports
interactions with a unique set of ECM proteins and sometimes soluble ligand
proteins in a cell type specific manner (Hynes, 1992). The integrins are
prominent membrane receptors for ECM components forming a transmembrane
link between the ECM and the actin cytoskeleton and transmit signals to
promote diverse cellular responses including adhesion, survival and
migration(Hynes, 1992). The physical connection between the actin cytoskeleton
and the ECM by integrins is critically important to provide traction at points of
cell-ECM adhesion. As more linkages take place, focal adhesion complexes are
formed, which are mature adhesion sites required for cell migration.
Importantly, breakage of this integrin-actin linkage is also essential for
disassembly and turnover of cell-ECM adhesions that permits the cell to migrate
(Ridley et al., 2003). Integrins on tumor cells contribute directly to adhesive
interactions of metastatic cells with different components of the tumor
microenvironment. For example, increased level of expression of integrin αvβ3 is
closely associated with increased metastasis (Felding-Habermann et al., 2002)

3
and cell migration (Brooks et al., 1994). In an additional example, the attachment
of ovarian cancer cells to peritoneal surface is primarily mediated by α5β1
integrin binding to fibronectin (Mitra et al., 2011).
Disintegrins are a family of small, cysteine-rich, naturally occurring
polypeptides found in snake venom (Gould et al., 1990). Many of them contain
Arg-Gly-Asp sequence that bind with high affinity to a subset of integrins on the
surface of normal and malignant cells and have the ability to inhibit the binding
of the natural ligand thus blocking normal integrin function (Scarborough et al.,
1993). Originally, Arg-Gly-Asp-disintegrins were characterized as platelet
aggregation inhibitors (McLane et al. 1998). As integrins have been
demonstrated to play a critical role for the proliferative, adhesive and migratory
properties of tumor cells, any disintegrin that can interfere with these processes
may possibly be used in the treatment of tumor growth and cancer metastasis.
Vicrostatin (VCN) is a sequence-engineered disintegrin, formed by grafting the C-
terminal tail of viperid snake venom disintegrin echistatin to the sequence of the
crotalid disintegrin contortrostatin (Minea et al., 2010). VCN has been shown to
target epithelial cells via αvβ3, αvβ5, and α5β1 integrin ligation, significantly
inhibiting their motility. Also, VCN disrupts the actin cytoskeleton, inhibits
human umbilical vein endothelial cells tube formation, and acts as a tumor anti-
angiogenic agent as illustrated in a human-in-mouse orthotopic xenograft breast
cancer model (Minea et al., 2010)

4
Monitoring the ability of VCN to alter the capacity of cells to migrate is
critical to aiding in the understanding of the mechanisms of VCN as an anti-
tumor / anti-angiogenic agent. The colloidal gold migration assay, also called
phagokinetic track assay, initially was used to investigate the patterns and
direction of migrating fibroblasts (Albrecht-Buehler, 1977), and has also been
adapted for the quantitative analysis of cell motility (Zetter BR, 1980). The
method involved in this assay is based on the nature of motile cells engulfing,
pushing to the sides, or collecting small particles on their bottom surface along
their moving path. In this method, first described by Albrecht-Buehler in 1977,
glass coverslips were coated with bovine serum albumin (BSA) and paved with
colloidal gold particles produced by reduction of chloroauric acid, HAuCl
4
‧3 H
2
O
(Albrecht-Buehler, 1977). Cells were then seeded onto the film-like gold layer
and as cells move, clear, particle-free phagokinetic tracks were formed, leaving a
permanent record of their migration. Quantification of cell migration was
achieved by using a computer-assisted image analysis program. One of the main
advantages of this technique over existing methods such a time lapse imaging is
that this assay requires only a standard light microscope, a standard digital
camera and freely available imaging software. In addition, this assay allows
analysis of comparison of multiple samples, using a method that is capable of
high-throughput screening. Although this method can provide insight on the
influence of different external, internal and physiological factors on cell
movement and migration, it can be also applied to other systems such as a study

5
of the effects pathogens have on the motility of infected cells. Studies of a wide
spectrum of cell types that utilized this assay include fibroblasts (Albrecht-
Buehler, 1977), neutrophils (Kawa et al., 1997), keratinocytes (Ando and Jensen,
1993, and endothelial cells (McAuslan et al., 1980).
Given that integrins are promising targets for development of cancer
therapy and VCN has shown integrin specificity as an antagonist of these
integrins, my aim was to establish a cost-effective cell migration assay to study
the anti-migratory effect of VCN now, as well as for future study of disintegrin
species with anti-migratory properties. The research will first focus on optimizing
the conventional colloidal gold migration method with the cancer cell lines we
currently investigate and then implement the advanced migration assay with
VCN to evaluate VCN potency in suppressing cancer cell motility in vitro. My
hypothesis is that VCN will inhibit the in vitro cell migration through binding to
specific integrins and show strong anti-tumor efficacy in human ovarian cancer
xenograft nude mouse model.

6
Materials and Methods
Cell culture
The human breast cancer cell line, MDA-MB-231, and ovarian cancer cell
lines, Hey and SKOV-3 were obtained from American Type Culture Collection
(ATCC, Rockville, MD). MDA-MB-231 was grown in Dulbecco’s modified Eagle’s
Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml
penicillin and 0.1 mg/ml streptomycin; Hey and SKOV-3 were maintained in
Roswell Park Memorial Institute medium (RPMI) supplemented with 10% FBS,
100 units/ml penicillin and 0.1 mg/ml streptomycin. Cells were incubated in a
humidified atmosphere containing 5% CO
2
at 37°C.
To subculture cell lines, cell media in each T75 flask was drained and 5 ml
phosphate buffered saline (PBS) was added to wash the flask. Subsequently, cells
were harvested by trypsinization with 5 ml of 10% trypsin stock (0.05% trypsin-
0.02% EDTA) in PBS for 5 minutes, followed by quenching in 5 ml of complete
media. Cells were resuspended in complete media and transferred to a new T75
flask containing fresh complete media in a specific ratio depending on the cell
line.
Reagents
The cytochalasin D was purchased from Calbiochem (San Diego, CA). The
bovine Fibronectin Solution was purchased from PromoCell (Heidelberg,
Germany). The rat tail type I collagen was purchased from BD Bioscience
(Franklin Lakes, NJ).

7
Expression and purification of VCN
VCN was recombinantly made and purified by the previously published
method described by Minea et al. (Minea et al. 2012). Briefly, the VCN sequence
was cloned into pET32a vector system downstream of Thioredoxin (Trx) using a
BglII/NcoI set of restriction enzymes and expressed on the Origami B (DE3) E. coli
strain. The transformant was grown, induced and then lysed in a microfludizer
(Microfluidics M-110 L, Microfluidics, Newton, MA). Centrifugation of the lysate
at 40,000 xg was used to remove insoluble cellular debris and collect the soluble
material. The soluble lysate containing Trx-VCN fusion proteins was then
proteolyzed by recombinant TEV protease overnight at room temperature. The
proteolyzed lysates were passed through a 0.22 mm filter, diluted 1:100 in
deionized H
2
O (DI H
2
O), ultrafiltered through a 50,000 MWCO cartridge
(Biomax50, Millipore) and then reconcentrated against a 5000 MWCO cartridge
(Biomax5, Millipore) using a tangential flow ultrafiltration device (Labscale TFF
system, Millipore).
Purification was accomplished by C-18 reverse phase high pressure liquid
chromatography using the standard elution conditions according to a previously
established method (Minea et al., 2010). The filtered lysates were processed as
described above and were loaded onto a Vydac C-18 column (218TP54,
Temecula, CA). A 10-minute wash/rinse (at 5 ml/min) of the column with an
aqueous solution containing 0.1%TFA was followed by elution with a linear
gradient (0–100%) over 150 min in a mobile phase containing 80% acetonitrile

8
and 0.1%TFA. VCN started eluting in 35% acetonitrile.
The purified VCN was initially characterized by sodium dodecyl sulfate
polyacrylamide gel electrophoresis to migrate to approximately M
r
7,000 under
reducing conditions and then tested for activity against platelet aggregation as
previously described by Minea et al. (Minea et al., 2012). A main function of
disintegrins is to bind to the activated platelet integrin αIIbβ3, thus inhibiting the
last step the aggregation of platelets, a process mediated by platelet integrin
αIIbβ3 (McLane et al., 2008).
Colloidal gold migration assay -96 well microplate format
The original protocol for the colloidal gold migration assay (Albrecht-
Buehler, 1977) as described was modified for this study as described below. All
solutions in experiments for making the gold-coated plate were filtered with
syringe filter of 0.2 μm cellulose acetate membrane (VWR International, Radnor,
PA) before use. Then, 96-well, polystyrene, flat-bottom, untreated microplates
(VWR International, Radnor, PA) were washed with DI H
2
O to remove the
impurities and the plates were covered or handled with an upward airflow at all
time to prevent contamination. After being dried, 0.1 ml of fresh prepared 1%
BSA was added to each well and allowed to incubate at room temperature for 30
minutes. The 1% BSA solution was then aspirated and the plates were dried at
60°C for 30 minutes.

9
The following reagents were mixed at room temperature in a 125 ml
Erlenmeyer flask: 20.8 ml of DI H
2
O, 12.0 ml of 36.5 mM Na
2
CO
3
, and 3.5 ml of
hydrogen tetrachloroaurate(III) (0.342g HAuCl
4
‧3 H
2
O dissolved in 50 ml DI
H
2
O). The flask was covered with foil to prevent evaporation and heated over a
Bunsen burner flame. The mixture was gradually heated while gently and
continually swirling the liquid. Rapid heating or vigorous shaking would result in
failure of the gold nanoparticles to precipitate out. The solution was heated until
boiling point and maintained in heat until large bubbles were observed.
Immediately, with the solution still at its boiling point, 3.6 ml of 0.1% w/v
formaldehyde was added with continued gentle swirling. During this time, the
solution would change from a faint yellow/clear, to gray, purple, deep purple
and eventually red wine or, preferably, rust color. This mixture was then
immediately transferred to the BSA-coated 96 well plates 0.1 ml, unless
otherwise specified, per well. The plates were allowed to stand at 37 °C for 30
minutes undisturbed and covered during which time gold nanoparticles were
deposited onto the bottom of the plate
After the particles had time to settle onto the bottom of the wells, the
density was evaluated under a light microscope. Based on visual assessment of
the gold density, if the concentration of the particle was insufficient, an
additional 0.05 to 0.1 ml of colloidal gold solution was added to the wells. If,
however, the concentration of gold nanoparticles was too high or showed a
heterogeneous coating or uneven density, the plates were discarded and the

10
production of plates was repeated. Once the plates were deemed acceptable
and the gold density was correct, the plates were stored at 4°C and sealed with
parafilm to prevent the wells from drying out.
Prior to beginning the migration assays, the plates were UV sterilized,
washed with 0.2 ml PBS 3 times and then the desired ECM components (0.02
mg/ml for fibronectin or 0.02 mg/ml for type I collagen) or other substrates
specified in the experimental design were placed onto the gold particle layer.
The ECM or other substrate solution with the plates were then incubated at 37°C
for 10 minutes before use or the plates were stored at 4°C until use.
Cells to be used in these assays were grow to 80-90% confluency in T75
tissue culture flask and were starved for 16 h. Before final use, the cells were
washed with 5 ml PBS, trypsinized with 5ml typsin, pelleted by centrifugation,
resuspended with 1-3ml of serum-free medium, and counted by hemocytometer.
Then, 8,000 cells were added to 1ml of conditioned medium and 0.1 ml of the
cell mixture was loaded into each well of the designated conditioned wells of the
ECM-covered and gold-coated plates that were pre-warmed at 37°C. The plates
were incubated at 37 °C with 5% CO
2
for up to 24 h depending on the cell line.
Following incubation of the cells in the experimental wells, migration of
the cells was evaluated by photographing randomly selected fields with an
Olympus E20N digital camera (5.0 MP acquisition; Olympus America, Melville,
NY) connected to the light microscope (Zeiss Axioplan-2 optical microscope).

11
Images were analyzed by digitally drawing lines of migration tracks and
quantitating the enclosed areas with the SimplePCI advanced imaging software
(Hamamatsu Corporation, Sewickley, PA). The computerized readouts from
SimplePCI were then standardized with a standard curve that was made by
measuring a series of areas on a hemocytometer to convert the Pixel unit into
metric unit. The level of cell migration was defined as average area cleared per
cell in square micrometer (μm
2
), which was calculated by the total area of
particles-free tracks generated by motile cells divided by the cell number in the
viewed field.
Statistical analysis
Statistical analyses of differences in cell migration between triplicate sets
of experimental conditions were performed using Microsoft Excel by unpaired t-
test. Confirmation of a difference in cell migration as statistically significant
requires rejection of the null hypothesis when the two-tailed p-value ＜0.05 with
the Student’s t-test.
In vivo efficacy of Oxiplex/VCN in an animal model of ovarian cancer
For in vivo efficacy studies, human ovarian cancer cells, OVCAR-3,
growing as a monolayer in culture were harvested with 0.05% trypsin-0.02%
EDTA, washed with PBS, and resuspended in complete medium. OVCAR-3 cells
were suspended in 4 ml PBS and 0.1 ml of the cell suspension (5 x 10
5
) was
injected intraperitoneally into 10 five-week-old female athymic nude mice
(Balb/c/nu/nu) weighing approximately 20 g each (Simonsen Labs, Gilroy, Calif.).

12
Each animal received 5 x 10
5
cells, and the tumors were allowed to implant for
19 days prior to treatment. The animals were maintained in a pathogen-free
condition and received sterilized commercial food and water ad libitum. After 19
days, groups of 5 animals each received either: intraperitoneal (IP) injections of 5
mg VCN dissolved in 1 ml Oxiplex gel weekly, or 1ml IP PBS injections weekly,
during the 28 days time period of the experiments. Oxiplex (Fizio Med, San Luis
Obisbo, CA) is a synthetic polymer compound of carboxymethyl cellulose and
polyethylene. It is featured by its high viscosity, absorbability and safety
established in spine surgery as a adhesion barrier to prevent post-surgical
adhesion (Kim et al., 2003; Liu et al. 2013). In this study, Oxiplex was used to
carry disintegrin VCN and to slowly release it over time. The sustained slow
release of VCN allowed long-lasting exposure of tumor to pharmacological
concentrations of the treatment.

13
Results
For the development of a platform that could be used in cell migration
assays for assessing the effectiveness of potential migratory inhibitors, we used
VCN as a functional disintegrin that antagonizes integrin-dependent migration.
We modified the colloidal gold migration assay (Albrecht-Buehler, 1977) that
was conventionally done on glass coverslips and developed a 96-well microplate
format. This minimized the volume of reagents, substrates and proteins used in
experiments and also, the quality, reproducibility and validity of the assay were
also ensured. Originally in the coverslip setup, it took 3 ml gold salt solution to
make a gold-coated glass coverslip and another 3 ml ECM solution to plate on
top of the gold layer. However, in 96-well format, an individual well with 0.32
cm
2
bottom area required as little as 0.04 ml of gold salt solution to cover the
bottom of the well and each well could be loaded with approximately 800 cells
(2,500 cells/cm
2
) spread out uniformly, which still is a sufficient cell number to
provide a statistically significant readout.
To adapt the conventional method to our study, the amount of gold salt solution
for making quality gold-coated plates was experimentally determined as shown
in Figure 1. (Panel A to C) 0.05, 0.1 and 0.3 ml of gold salt solution were added to
the plate respectively, and allowed to settle to make different densities of gold
particle layer. By the optical microscope with 100x magnification, it was
observed that 0.05 ml gold salt solution per well (panel A) provided a relatively
loose gold layer compared to 0.1 ml (panel B) and 0.3 ml of gold salt solution

14
(panel C). It should be noted that the optimal density is varied with cell size and,
if the concentration of gold particle is too high, it hampers the ability of cells to
move; while if the concentration of gold particle is too low, it limits the ability to
delimit an accurate migration track. Here, we chose highly metastatic human
cancer cell lines, MDA-MB-231, Hey and SKOV-3 cells, as models. Cells were
cultured, harvested as described in Materials and Methods and then seeded
onto the series of densities of gold layer with 0.02 mg/ml fibronectin covered on
top of the gold layer and then incubated at 37 °C 5% CO
2
. In approximately 1 hr,
Hey cells attached to the gold layer, took up gold particles and some of them
began migration (Fig. 1, panel D) and reached maximal level of migration around
10 hr (Fig. 1 panel E and F), when visualized under an optical microscope. Via
100x magnification (Fig. 1 panel F), it could be clearly seen that the Hey cells
moved on the gold layer, pushed the gold particles aside and left gold
nanoparticle-free paths behind. Also, the gold particles were engulfed by the
cells and accumulated inside their cytosol, which made the cells more visible to
be observed after phagokinesis had taken place. To better capture the whole
picture in each condition and to mark multiple cell migration tracks for statistical
analysis, we later used 12.5x magnification to photograph the cell migrations as
Figure 3. That rendered around 25 to 40 cell migration tracks on a single image.
During the time periods of the assay, cell proliferation was not observed, as the
doubling time of Hey is about 16 hr (Shahabi et al., 2010), and the presence of
0.01 mg/ml mitomycin C did not block the migration (data not shown).

15
Figure 1

Examples of colloidal gold-coated surface and the migration tracks. Shown here
are examples of 96 well microplate coated with various densities of gold
nanoparticles (Panel A, B and C). Human ovarian cancer cell line, Hey, cultured in
complete medium in a tissue culture flask to 90% confluence and then serum-
starved for 16 h, were lifted off by trypsinization, pelleted by centrifugation,
resuspended, and counted by hemocytometer. White arrows mark Hey cells in
their final location. Approximately 800 cells per well were seeded on each pre-
prepared fibronectin-coated gold salt covered well of 96 well assay plate in RPMI
plus 1% FBS and then incubated at 37 °C 5% CO
2
for 10 h. Pictures were taken at
25x (Panel D and E), 50x (Panel F) or 100x magnification (Panel A, B and C). Size
bar corresponds to 100 μm in length.

16
The MDA-MB-231 and Hey cells produced long linear migration tracks in a
dose-dependent manner as determined by FBS concentration and their cell
migration levels, were calculated, using the SimplePCI imaging program as
described in Materials and Methods (Fig. 2). Cell migrations were enhanced with
the increase of FBS concentration. In 10% FBS by 10 hr, cell migration had
maximized to around 12,500 μm
2
for MDA-MB-231 and 14,500 μm
2
for Hey. In
contrast, no linear tracks were observed on SKOV-3 either at the 1 hr or 10 hr
time point in 0 to 10 % FBS conditions. As mentioned above, the appropriate
density of gold layer varied with the size of cells. Since the size of SKOV-3 is
smaller than MDA-MB-231 and Hey cells, the working gold density for the two
cell lines was likely too much for SKOV-3 to migrate. Hence, the density of gold
particles needs to be optimized for SKOV-3 individually to ease the physical
constraint to SKOV-3 cells created by excessive gold deposit. Other factor that
could limit SKOV-3 cell migration was improper ECM component used in the
assay. The ECM proteins as substratum for cell migration are cell type-
dependent and the fibronectin we plated was not able to initiate SKOV-3 cell
migration. It was reported that, for example, in human keratinocytes, cells were
most motile when seeded on type I collagen (Li et al. 2001), but they would be
otherwise inhibited if seeded on laminin-5 (O’Toole et al. 1997). The
phenomenon of the specificity of cell migration to ECM component is linked to
integrin expression and integrin binding specificity to various ECM proteins in
each cell line. SKOV-3 was shown to have highest binding specificity to type I

17
collagen than other ECM (Cannistra et al. 1995). However, we tried seeding
SKOV-3 on top of type I collagen-covered surface, but it still showed no
improvement on cell motility (data not shown). To initiate successfully SKOV-3
cell migration, other type of ECM or combinations of ECM components needed
to be tested.

18
Figure 2

Quantitative evaluation of MDA-MB-231, Hey and SKOV-3 cell migration. MDA-
MB-231, Hey and SKOV-3 cells cultures were prepared as described in Materials
and Methods. Approximate 800 cells per well were seeded onto the fibronectin-
covered plate in assigned medium and then incubated at 37 °C 5% CO
2
for 10 h.
Triplicates photographs of three random fields from three wells were taken and
the cell migration was computed by measuring the average area cleared per cell
using SimplePCI imaging software. Error bars show the standard deviation (SD) of
three fields.

19
To investigate the potency of VCN as an integrin-antagonist to inhibit cell
migration, we tested the effect of VCN on MDA-MB-231 and Hey cell migration.
In these experiments, cells were suspended in growth media with or without
VCN at a series of concentrations and the fungal metabolite cytochalasin D, a
potent inhibitor of actin polymerization, was used as a positive control at a
concentration of 400 nM. The cells were then subjected to the migration assays.
We found that VCN attenuated MDA-MB-231 and Hey cell migration on a
fibronectin-covered plate in a dose-dependent manner (Fig. 3). To measure
effectiveness of VCN in inhibiting cell migration, phagokinetic tracks were
quantitated for MDA-MB-231 (Fig. 4) and Hey (Fig. 5) cells. The anti-migration
potency of VCN showed statistical significance at a concentration of 100 nM and
above when the cells were seeded on a fibronectin-covered surface.
Nevertheless, if the cells were seeded on a type I collagen-covered surface, VCN
did not have evident effect on cell migration (Fig. 4 and Fig. 5). Meanwhile, cell
migration was drastically abolished in all conditions with addition of 400 nM
cytochalasin D. The differences of VCN effects between fibronectin- and type I
collagen-covered surface can be explained by the specific binding affinity of VCN,
which is specific to αvβ3, α5β1 and αvβ5 integrins. αvβ3 and α5β1 integrins are
known to be the integrins that bind to fibronectin but no collagen-binding
integrins (α1β1, α2β1 and α3β1) are targeted by VCN. Therefore, when VCN
functionally blocked these types of integrins in cells, the cells on fibronectin-
covered surface would not execute integrin-dependent cell migration via these

20
VCN-ligated integrins. On the other hand, when cells were plated on top of the
type I collagen-covered surface, cell migration was not interfered with by VCN
treatment since the cells might use other types of integrins to form focal complexes to
migrate.

21
Figure 3
Hey and MDA-MB-231 cell migration in the presence of VCN. Hey and MDA-
MB-231 cells in culture were trypsinized, washed, and resuspended in RPMI or
DMEM with the indicated concentrations of VCN or cytochalasin D (CytoD).
Approximately 800 cells per well were plated on fibronectin-coated plate in
RPMI or DMEM with 1% FBS. Photos were taken with 12.5x magnification after
10 h incubation. Representative photographic images of the migration tracks
under each of the conditions indicated. Size bar corresponds to 100 μm in length.

22
Figure 4
Inhibition of Hey cell migration by VCN. Hey cell migration assays were
prepared as described in Materials and Methods. The figure compares cell
motility of 10 hr migration assays with a series of concentrations of VCN or 400
nM CytoD as control in RPMI with 1% FBS. Triplicate photographs of three
random fields from three wells were taken and the cell migration was computed
by measuring the average area cleared per cell as described in Materials and
Methods. Error bars show the SD of three fields. Significant differences from the
control are indicated: *P < 0.05.

23
Figure 5
Inhibition of MDA-MB-231 cell migration by VCN. MDA-MB-231 cell migration
assays were prepared as described in Materials and Methods. The figure
compares migration activities of 10 hr migration assays with the addition of
series of concentrations of VCN or 400 nM CytoD as control in DMEM with 1%
FBS. Triplicates photographs of three random fields from three wells were taken
and the cell migration was computed by measuring the average area cleared per
cell. Error bars show the SD of three fields. Significant differences from the
control are indicated: *P < 0.05.

24
Since VCN was a potent inhibitor in vitro of human ovarian cancer cell,
Hey, migration, we hypothesized that VCN would be effective in limiting the
dissemination of ovarian cancer and possibly slow growth of the primary tumor.
A human ovarian cancer xenograft mouse model was used to study the efficacy
of VCN in the highly metastatic ovarian carcinoma OVCAR-3 cell line. Ten female
nude mice were inoculated with ovarian carcinoma (5 x 10
5
) OVCAR-3 cells. After
allowing the tumor to grow for 19 days, the mice were treated weekly with
intraperitoneal injection of 5 mg VCN that was dissolved in 1 ml Oxiplex gel,
which is for human use in surgery to prevent post-surgical adhesion formation
available in nearly 70 countries, including those in the European Union, Australia,
Canada, Brazil, Mexico and South Korea but not USA. Here, Oxiplex gel was
utilized to carry VCN and slowly release it. Animals receiving saline injection
were used as controls. The results showed that the saline-treated control mice
had tumors grow and disseminate to stomach and peritoneum (Fig. 6, top panel).
In contrast, the VCN-treated mice had almost no visible tumors (Fig. 6, bottom
panel). In the survival analysis, VCN was found to greatly increase the survival of
the animals compared to the control mice (Fig. 7). Our preliminary animal study
demonstrated that VCN showed promising effects on not only tumor spread but
also survival of the mice.

25
Figure 6

Macroscopic evaluation of tumor burden. Each animal was injected with 5x10
5

OVCAR-3 cells intraperitoneally (IP) and the cells were allowed to implant for 19
days prior to treatment. Top: Female nude mouse 32 days after IP inoculation
with live OVCAR-3 cells. Bottom: Mouse with same inoculation, with weekly IP
injections of VCN delivered in a slow-release gel. The treatment group displayed
no visibly detectable tumor growth in comparison to the control group. Arrows
show tumor spread to stomach and peritoneum.

26
Figure 7
Kaplan-Meier curve for OVCAR-3 tumor model. Five animals from the 2 groups
were used for the survival study (N=10). All mice were inoculated with the
OVCAR-3 ovarian carcinoma cells and allowed to grow for 19 days before
treatment started. The treatment regimen of 5 mg VCN in slow-release gel
delivered weekly IP on Day 1 and maintained for the 28 day experiment to see if
the treated animals would out live control animals. The animal data shows
increased survival in VCN treated groups compared to the control.

27
Discussion
Oncology as a therapeutic approach has a poor record in clinical drug
development with three times lower success rates than for cardiovascular
diseases (Kamb, 2005). The inferior performance of many investigational drugs
implies that the standard models for screening potential compounds are
unsatisfactory and defective. Partly it is because the majority of preclinical
compounds that enter clinical trials for cancer therapy have been selected on the
basis of cytotoxicity profile (Kamb et al., 2007). Yet, most advanced cancer
patients die because of the highly migratory nature of cancer cells. In fact, it is
now well acknowledged that cell migration plays critical roles in tumor invasion
and cancer metastasis (Hanaban and Weinberg, 2000). Therefore, investigators
today have to develop anti-migratory agent as an alternative to, or in
combination with, treatments designed to kill cancer cells.
Given the fundamental importance of in vivo cell motility, various in vitro
methodologies have been established to characterize this phenomenon more
easily and to allow insightful investigation on the effects of endogenous or
exogenous substances on cell migration. In vitro assays are generally used to
provide preliminary information because in vivo models are usually more difficult
and time- and money-consuming to perform. Besides that, it is also more
complicated and difficult to quantify in in vivo tests. This is why in vivo tests are
mainly used as the ultimate stage to confirm information provided by in vitro
assays. Here we describe and analyze the pro and cons of two other methods as

28
well as the colloidal gold migration assay that are available to monitor and to
characterize the migratory behavior of cancer cells. Boyden chamber assay, or
transmembrane assay, first described by Boyden for the analysis of leukocyte
chemotaxis (Boyden, 1962), is one of the most common assays used to study cell
migration. In general, this assays consists of two medium-filled compartments
separated by a microporous membrane filter, often coated with ECM proteins.
Cells are seeded in the upper chamber and are allowed to migrate through the
porous filter into the lower chamber, in which chemoattractant or migratory
inhibitor is present. After incubation for a period of time, cells that invade and
migrate through the membrane are stained and counted to determine cell
migration level. This assay is advantageous by its compatibility with both
adherent and non-adherent cells and possible use of chemotactic gradient in the
assay. However, several issues in this assays are that 1) in vivo cell migration
does not need the membrane filter structure to take place; 2) size of pore used
in the assay directly affects the ability of cells to pass through; 3) the
concentration gradient of the chemoattractant is variable and unknown.
Traditionally, this assay has been an endpoint assay for the fact that the cells
must be dissociated from the membrane to measure cell migration. Lately, the
cell counting problem is solved by using a commercially available “light tight”
membrane that can block the transmission of fluorescent detection light
(Bernhagen et al., 2007). Thus, fluorescently labeled cells are detectable now in
real time for kinetic assay. The second assay is the Scratch assay, also known as

29
wound healing assay. It is introduced as an easy and low-cost method to quantify
cell migration (Liang et al., 2007) in an artificial wound healing process. Basically,
the assay involves creating a cell-free scratch in a cell monolayer and capturing
photographic images during the assay to record the change of the scratched
region where cells migrate in. Results from this assay are inconsistent due to the
fact that scratch was made by a pipette tip. More recently, automated robotic
pin tool that makes reproducible scratches are introduced to make the assay
more consistent and compatible with a high-throughput screening setup (Yarrow
et al., 2004). The advantages of scratch assay are that it is simple to operate and
cell migration can be observed in real time. Disadvantages are: 1) the process of
making scratch also damages the cells and the ECM underneath the cells; 2) the
damage to the cells at the edge of the scratch can lead to dysfunction in
migration and may cause release of undefined factors from damaged cell into
the media and 3) gradients of soluble factors and chemoattractant effects
cannot be studied in this assay. The major advantages of the colloidal gold
migration assay used in this study are its sensitivity and reproducibility. Area of
migration track can be accurately measured and large quantities of assay plates
can be easily manufactured from the same batch of gold salt solution to ensure
the quality of plates are consistent. Also, the selection of ECM as substratum
based on specificity of member receptor is feasible and the phagokinetic assay
requires relatively low numbers of cells, which is particularly useful for cells that
are difficult to grow in large numbers.

30
Drug delivery to tumors is necessary for chemotherapy to be effective,
especially in ovarian cancer, which is predominantly confined to the peritoneal
cavity, so the strategy of localized IP chemotherapy is urgently needed
(Dvoretsky et al., 1988). In this study, Oxiplex gel that had been approved for
human use in Europe and Australia for prevention of post-surgical adhesion
(Rodgers et al., 2003) was utilized as delivery vehicle to achieve the benefits of
high-concentration local peritoneal treatment as well as sustained drug delivery.
Also, in the management of ovarian cancer, use of Oxiplex has the advantage of
preventing the formation of post-surgical adhesions. The in vivo ovarian cancer
mouse model in this study showed that Oxiplex/VCN IP injections prevented the
dissemination of primary tumor, decreased tumor growth and prolonged survival
in study animals. To further explore this highly translatable research for
development of ovarian cancer therapy, additional mouse models mimicking
ovarian cancer recurrence are in progress in our laboratory. It is hoped that
theses proof-of-concept studies will aid in the development of both a novel
therapeutic agent as well as a novel drug delivery system.
Integrins have a large role in both angiogenesis (Contois et al., 2009) and
cancer migration (Hood and Cheresh, 2002) during tumor progression. Hence,
screening and selecting therapeutic integrin antagonists are gaining popularity
for potential anti-cancer agents (Folkman, 2007). In this study, we demonstrate
that a novel and recombinantly produced disintegrin, VCN, has the evident anti-
migratory property in human breast cancer and ovarian cancer cell models as

31
discussed in this report. Additional mechanistic studies are currently underway
in our laboratory in an effort to better understand the signaling, and possibly
therapeutic, differences between disintegrins and other soluble integrin ligands.

32
References
Albrecht-Buehler G. The Phagokinetic tracks of 3T3 cells. Cell 11:395–404,
1977
Brooks PC, Clark RA, Cheresh DA. Requirement of vascular integrin αvβ3 for
angiogenesis. Science 264:569–571, 1994
Ando Y, Jensen PJ. Epidermal growth factor and insulin-like growth factor I
enhance keratinocyte migration. J. Invest. Dermatol. 100:633–639, 1993
Bernhagen J, Krohn R, Lue H, Gregory JL, Zernecke A, Koenen RR, Dewor M,
Georgiev I, Schober A, Leng L, Kooistra T, Fingerle-Rowson G, Ghezzi P,
Kleemann R, McColl SR, Bucala R, Hickey MJ, Weber C. MIF is a noncognate
ligand of CXC chemokine receptors in inflammatory and atherogenic cell
recruitment. Nature Medicine 13:587–596, 2007
Boyden SV. The chemotactic effect of mixtures of antibody and antigen on
polymorphonuclear leucocytes. J. Exp. Med. 115:453–466, 1962
Cannistra SA, Ottensmeier C, Niloff J, Orta B, DiCarlo J. Expression and
function of β1 and αvβ3 integrins in ovarian cancer. Gynecol. Oncol. 58:216–
225, 1995
Contois L, Akalu A, Brooks PC. Integrins as “functional hubs” in the regulation
of pathological angiogenesis. Semin. Cancer Biol.19:318–328, 2009
Dvoretsky PM, Richards KA, Angel C, Rabinowitz L, Beecham JB, Bonfiglio TA,
Survival time, causes of death, and tumor/treatment-related morbidity in 100
women with ovarian cancer. Hum. Pathol. 19:1273–1279, 1988
Felding-Habermann B, Fransvea E, O ’Toole TE, Manzuk L, Faha B, Hensler M.
Involvement of tumor cell integrin αvβ3 in hematogenous metastasis of
human melanoma cells. Clin. Exp. Metastasis 19:427–436, 2002

33
Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat. Rev.
Drug Discov. 6:273–286, 2007
Friedl P and Wolf K. Plasticity of cell migration: a multiscale tuning model. J.
Cell. Biol. 188:11–19, 2009
Gould RJ, Polokoff MA, Friedman PA, Huang TF, Holt JC, Cook JJ, Niewiarowki
S. Disintegrins: a family of integrin inhibitory proteins from viper venoms. Proc.
Soc. Exp. Biol. Med. 195:168–171, 1990
Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 7:57–70, 2000
Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat. Rev.
Cancer 2:91–100, 2002
Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell
69:11–25, 1992
Kamb A. What’s wrong with our cancer models? Nat. Rev. Drug Discov. 4:161–
165, 2005
Kamb A, Wee S, Lengauer C. Why is cancer drug discovery so difficult? Nat.
Rev. Drug Discov. 6: 115–120, 2007
Kawa S, Kimura S, Hakomori S, Igarashi Y. Inhibition of chemotactic motility
and trans-endothelial migration of human neutrophils by sphigosine 1-
phosphate. FEBS Lett. 29: 196–200, 1997
Kim KD, Wang JC, Robertson DP, Brodke DS, Olson EM, Duberg AC, BeDebba
M, Block KM, diZerega GS. Reduction of radiculpathy and pain with
Oxiplex®/SP gel following laminectomy, laminotomy and discectomy: a pilot
clinical study. Spine 28: 1080–1087, 2003
Li W, Nadelman C, Henry G, Fan J, Muellenhoff M, Medina E, Gratch NS, Chen
M, Han J, Woodley D. The p38-MAPK/SAPK Pathway is required for human

34
keratinocyte migration on dermal collagen. J. Invest. Dermatol. 117:1601–
1611, 2011
Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and
inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2:329–
333, 2007
Liu ZC, Li Y, Zang Y, Cui G, Sang HX, Ma ZS, Kong L, Lei W, Wu ZX. Clinical
assessment of a CMC/PEO gel to inhibit postoperative epidural adhesion
formation after lumbar discectomy: a randomized, control study. Arch. Orthop.
Trauma Surg. 133:295 –301, 2013
McAuslan BR, Hannn GN, Reily W. Characterization of an endothelial cell
proliferation factor from cultured 3T3 cells. Exp. Cell. Res. 128:95–101, 1998
McLane MA, Marcinkiewicz C, Vijay-Kumar S, Wierzbicka-Patynowski I,
Niewiarowski S. Viper venom disintegrins and related molecules. Proc. Soc.
Exp. Biol. Med. 219:109–119, 1998
McLane MA, Joerger T, Mahmoud A. Disintegrins in health and disease. Front.
Biosci. 13:6617–6637, 2008
Minea RO, Helchowski CM, Rubino B, Brodmann K, Sweson SD, Markland FS
Jr. Development of a chimeric recombinant disintegrin as a cost-effective anti-
cancer agent with promising translational potential. Toxicon 59:472–486, 2012
Minea RO, Helchowski CM, Samuel JZ, Costa FK, Swenson SD, Markland FS Jr.
Vicrostatin – an anti-invasive multi-integrin targeting chimeric disintegrin with
tumor anti-angiogenic and pro-apoptotic activities. PLoS 3:e10929, 2010
Mitra AK, Sawada K, Tiwari P, Mui K, Gwin K, Lengyel E. Ligand-independent
activation of c-Met by fibronectin and α5β1-integrin regulates ovarian cancer
invasion and metastasis. Oncogene 31:1566–1576, 2011

35
O ’Toole EA, Marinkovich MP, Hoeffler WK, Furthmayr H, Woodley DT.
Laminin-5 inhibits human keratinocyte migration. Exp. Cell. Res. 233:330–339,
1997
Ridley AJ, Schwartz MA, Burridge K, Firtel RA, Ginsberg MH, Borisy G,
Parsons JT, Horwitz AR. Cell migration: integrating signals from front to back.
Science 5:1704–1709, 2003
Rodgers KE, Robertson JT, Espinoza T, Oppelt W, Cortese S, diZerega GS, Berg
RA. Reduction of epidural fibrosis in lumbar surgery with Oxiplex adhesion
barriers of carboxymethylcellulose and polyethylene oxide. Spine. 3:277–283,
2003
Sahai E. Mechanisms of cancer cell invasion. Curr. Opin. Genet. Dev. 15: 87–96,
2005
Scarborough RM, Rose JW, Naughton MA, Phillips DR, Nannizzi L, Arfsten A,
Campbell AM, Charo IF. Characterization of the integrin specificities of
disintegrins isolated from American pit viper venoms. J. Biol. Chem. 268:1058–
1065, 1993
Shahabi S, Yang CP, Goldberg GL, Horwitz SB. Epothilone B enhances surface
EpCAM expression in ovarian cancer Hey cells. Gynecol. Oncol. 119:345–350,
2010
Yarrow JC, Perlman ZE, Westwood NJ, Mitchison TJ. A high-throughput cell
migration assay using scratch wound healing, a comparison of image-based
readout methods. BMC Biotechnol. 4:21–29, 2004
Zetter BR. Migration of capillary endothelial cells is stimulated by tumour-
derived factors. Nature 285:41–43, 1980

Abstract (if available)

Abstract Cell migration underlies tissue formation, maintenance and regeneration as well as pathological condition such as cancer invasion and metastasis. The integrin family of cell adhesion proteins promotes the attachment and migration of cells on the surrounding extracellular matrix (ECM). Through signals transduced upon integrin ligation by ECM proteins, this family of proteins plays key roles in regulating tumor growth, angiogenesis as well as metastasis. During the last 15 years, the Markland laboratory has isolated and recombinantly produced several disintegrin species from southern copperhead snake venom, which act as integrin antagonists with the ability to disrupt integrin functions through direct binding. In this study, I examined the anti-migratory effect of Vicrostatin (VCN), a rationally designed and sequence-engineered disintegrin, with both in vitro quantifiable cell migration assay and in vivo human ovarian cancer xenograft mouse model. The results show that VCN at 100 nM and above significantly inhibits in vitro MDA-MB-231 and Hey cell migrations. In separate studies, it was shown in the lab that 5 mg VCN in slow-release gel by weekly intraperitoneal injection prevents in vivo tumor dissemination and increases host survival. These findings underscore the potential of VCN in anti-metastatic and anti-tumorigenic action as a novel treatment for breast and ovarian cancer.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Effect of vicrostatin on integrin based signaling molecules in cancer

PDF

Mechanistic studies of the disintegrin contortrostatin and characterization of the recombinant protein vicrostatin

PDF

Effect of vicrostatin (an integrin based therapy) on canine osteosarcoma

PDF

In vivo study with a novel liposomal formulation of a recombinant ADAM disintegrin-like domain showing both anti-angiogenic and tumor growth inhibition activities

PDF

Studies on the role of a novel protein, TMEM 56 in tumorigenic growth for MCF-7 cells

PDF

Interleukin-11: a study of its effects in glioblastoma multiforme

PDF

Examining perillyl alcochol in glioblastoma treatment and relating its effect to endoplasmic reticulum stress response to further advance its usage in combination treatment with dimethyl-celecoxib

PDF

Enhanced Burkitt 's lymphoma cell killing by the combination treatments of bortezomib with celecoxib and 2,5-dimethyl-celecoxib (DMC)

PDF

Wnt/β-catenin/p300 induced transcription is critical for the differentiation and maintenance of Paneth cells

PDF

Pten deletion in adult pancreatic beta-cells induces cell proliferation and G1/S cell cycle progression

PDF

Bone marrow derived mesenchymal stem cells promote survival and drug resistance in tumor cells

PDF

Developing a bioluminescence tracking system targeting on tumor cells and T cells for cancer immunotherapy

PDF

Construction of plasmids for constitutive and induced expression of secreted alkaline phosphatase in mammalian ovarian carcinoma cells

PDF

The mechanism by which extracellular Hsp90α promotes cell migration: implications in wound healing and cancer

PDF

Creating a multiple micrornia expression vector to target GRP78, an ER chaperone and signaling regulator in cancer

PDF

Exploration of the roles of cancer stem cells and survivin in the pathogenesis and progression of prostate cancer

PDF

Generation and characterization of fully human anti-CD19 chimeric antigen receptor T (CAR-T) cells for the treatment of hematologic malignancies

PDF

Snake venom proteins identification by nano high performance liquid chromatography tandem mass spectrometry

PDF

Tri-specific T cell engager immunotherapy targeting tumor initiating cells

PDF

Characterization of a fragment in secreted Hsp90α with potential therapeutic benefits in wound healing and cancer

Asset Metadata

Creator Chen, Chien-Yu (author)

Core Title Quantitative evaluation of the effectiveness of an integrin antagonist, vicrostatin, in cell migration

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 07/23/2013

Defense Date 06/04/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cell migration,disintegrin,integrin,Metastasis,OAI-PMH Harvest,vicrostatin

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Markland, Francis S. (committee chair), Declerck, Yves A. (committee member), Tokes, Zoltan A. (committee member)

Creator Email chen539@usc.edu,rabultrapi@yahoo.com.tw

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-298355

Unique identifier UC11294641

Identifier etd-ChenChienY-1831.pdf (filename),usctheses-c3-298355 (legacy record id)

Legacy Identifier etd-ChenChienY-1831-0.pdf

Dmrecord 298355

Document Type Thesis

Format application/pdf (imt)

Rights Chen, Chien-Yu

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA