[CANCERRESEARCH54, 4993-4998, September15, 19941
Contortrostatin,
Metastatic
a Snake Venom Disintegrin, Inhibits 1J@Integrin-mediated
Melanoma
Cell Adhesion and Blocks Experimental
Human
Metastasis1
Mohit Trikha, Yves A. De Clerck, and Francis S Markland2
Department of Biochemistry and Molecular Biology and University of Southern California Norris Comprehensive
Medicine, Lcs Angeles, California 90033
ABSTRACT
Disintegrins are Arg—Gly—Asp-containing proteins that inhibit integrin
mediated cell-cell and cell-matrix interactions. We have purified a disin
tegrin, contortrostatin,
from Agkistrodon
contortrir
contortrir
snake
venom that is a potent inhibitor of human metastatic melanoma (M24
met) cell adhesion to extracellular matrix proteins. Contortrostatin inhib
its M24 met cell adhesion to type I collagen, vitronectin, and fibronectin
with 50% inhibitory
concentration
values of 20, 75, and 220 aM, respec
lively. Contortrostatin does not significantly inhibit adhesion of M24 met
cells to laminin. ‘@‘I-labeled
contortrostatin binds to M24 met cells in a
saturable
and displaceable
manner.
Scatchard
analysis
indicates
that
there are two binding sites for tasI@labeledcontortrostatin on the surface
ofthese cells. High affinity binding has a Kd of3 tiMwith 165,000 sites/cell;
@
low affinity binding has a Kd of 60 aM with 500,000 sites/cell. Immobilized
contortrostatin
can support adhesion of M24 met cells; this binding is
blocked by a monoclonal antibody to the
integrin subunit and by an
antibody to the fibronectin receptor a@[email protected] anti-vifronectin
receptor
(a@@35)monoclonal
antibody
which
blocks
adhesion
of M24 met cells to
immobilized vitronectin does not block binding of M24 met cells to
immobilized contortrostatin.
In an in vivo experimental metastasis model
system, contortrostatin
at 20 pg and 100 pg inhibits lung colonization of
M24 met cells (5 x 1O@),injected in the tall vein of acid mice, by 51 and
73%, respectively. We conclude that contortrostatin is a potent inhibitor
of fi@integrin-mediated
M24 met cell adhesion in vitro and that it also
inhibits lung colonization in vivo.
INTRODUCTION
Integrins are an important class of cell surface receptors that are
critically involved in cell-cell and cell-matrix interactions (i). All
integrins are a/j3 heterodimers that require metal ions for noncovalent
association of their subunits (1). A recent review by Smyth et a!. (2)
indicates that there are at least 8 a and 15 13 subunits (2). The a
subunits vary in size between Mr i20,000 and 180,000 and the (3
subunits
range from Mr 90'000
to i 10,000.
The (3 subunits
Cancer Center, University of Southern California School of
specific marker for most malignant melanoma cells (3, 5—7).Quali
tative and quantitative changes in the fibronectin receptor, a531 , are
also involved in the proliferative response of quiescent human
melanoma cells (4, 8).
Several classes of integrin receptors recognize an RGD sequence
present in ECM proteins (i). Studies during the past few years have
demonstrated that RGD-containing peptides inhibit binding of tumor
cells to the ECM by effectively competing with ECM proteins for
integrin receptor sites (1). Recently small proteins containing an RGD
sequence have been purified from the venom of Crotalidae and
Viperidae snake species (9—18).These disulfide-rich proteins, called
disintegrins, are potent inhibitors of platelet aggregation. They bind to
the integrin receptor GPIIbIHIa (a11@j33),thereby inhibiting the final
common pathway of platelet aggregation, the binding of fibrinogen.
The concentration of disintegrins necessary to inhibit GPIIbIIIIa
mediated platelet aggregation is 1000—2000 times lower than that
required for small linear RGD peptides (16, 17).
We have previously reported the purification and characterization
of two disintegrins (18). Contortrostatin was purified from the venom
ofAgkistrodon contortrix contortrix and multisquamatin was purified
from the venom of Echis multisquamatus. Contortrostatin and mul
tisquamatin are potent inhibitors of GPIIb/HIa-mediated human plate
let aggregation with IC50 values of 49 and 97 n@i,respectively (18).
Contortrostatin4 and other disintegrins (19—21)have significant po
tential as antithrombotic agents for use in thrombolytic therapy.
In view of the effectiveness of contortrostatin as a f3@integrin
antagonist in vitro and its potent antithrombotic activity in vivo, we
investigated its efficacy as an inhibitor of (3@integrin-mediated inter
actions. In this publication we report that contortrostatin is an effec
tive inhibitor of (3@integrin-mediated M24 met cell adhesion in vitro
and that it blocks experimental metastasis of these melanoma cells
in vivo.
pair with
different a subunits, resulting in varying specificities toward ECM3
proteins (i, 2). The interaction of both normal and malignant cells
with ECM components such as fibronectin, vitronectin, types I and IV
collagens, and laminin is mediated by integrins (i, 2). Integrins have
also been shown to be involved in tumor metastasis (1—4). For
example, the vitronectin receptor (3) and the fibronectin receptor (4)
have been shown to be involved in the development of the metastatic
phenotype of invasive melanoma. The vitronectin receptor serves as a
MATERIALS
Materials.
AND METHODS
M24 met cells were a generous gift from Dr. Ralph Reisfeld
(The Scripps Research Institute, La Jolla, CA)(22). M24 met cells were grown
in RPMI
containing
prior to reaching
10% serum
confluency
and 5 mM glutamine.
and all experiments
Cells
were
were performed
passaged
within
10—15
passages. The CellTiter 96 kit used for the cell adhesion assay was purchased
from Promega (Madison, WI). Human extracellular matrix proteins were
purchased
from GIBCO-BRL
(Gaithersburg,
MD) or Promega. The rat j3@
integrin monoclonal antibody, mAb 13 (23), was a kind gift from Des. Steven
Received 2/3/94; accepted 7/20/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
I This
work
was
supported
by NIH
Department
of Health
and
Human
Services
whom
requests
for
reprints
should
be
addressed,
at
University
of
abbreviations
used
are:
ECM,
extracellular
matrix;
RGD,
Southern
Arg—Gly—Asp;
antibody;
murine
monoclonal
antibodies
a2 (P1E6),
a3
murine 133integrin monoclonal antibody (RUU-PL 7F12) was purchased from
Becton Dickinson (San Jose, CA). Anti-vitronectin receptor murine mono
California School of Medicine, Cancer Research Laboratory #106, 1303 North Mission
Road, Los Angeles, CA 90033.
3 The
a5@1, rabbit polyclonal
(P1B5), a5 (P1D6), and aj3@(P1F6); and a rabbit polyclonal antibody (a,j33,
135)
thatrecognizes
t@j33
andaj35werepurchased
fromGIBCO-BRL
The
Grants
CA54861 (F. S. M., Y. A. D.) and CA42919 (Y. A. D).
2 To
K. Akiyama and Kenneth M. Yamada (NIH). The anti-fibronectin receptor,
clonal antibodies P302 (anti-crj35) and LM609 (anti-aj33)
were a kind gift
from Dr. David Cheresh (The Scripps Research Institute) (24). Dynatech
GP,
glycoprotein; IC5@,,
50% inhibitory concentration; M24 met, human metastatic melanoma;
HPLC, high pressure liquid chromatography; ESI, electrospray ionization; PE, pyridyl
ethylated; PBS, phosphate-buffered saline (2.7 [email protected] inst KH2PO4-138mt.i NaC1-
immunolon II plates, purchased from Fisher Scientific (Pittsburgh, PA), were
used for cell adhesion assays. Fetal bovine serum was purchased from Irvine
8.1 mr@iNa2HPO4, pH 7.25); BSA, bovine serum albumin; SFM, serum-free medium;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamidegel electrophoresis;mAb,
monoclonal antibody.
4 F.
S.
Markland,
G.
S.
Friedrichs,
M.
Trikha,
and
B.
R.
Lucchesi,
manuscript
preparation.
4993
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in
CONTORTROSTATIN
INHIBITS
MELANOMA
Scientific (Santa Ma, CA) and RPMI was from GIBCO-BRL. A Dynatech
enzyme-linked immunosorbent assay plate reader was used for the cell adhe
ADHESION
AND
METASTASIS
purchased from Charles River Laboratories (Wilmington, MA). All other
achieved. All experiments utilizing ‘@I-labeledcontortrostatin were performed
within 3 weeks of iodination.
Scatchard Analysis. M24 met cells (5 X 10@/100 p1) or platelet-rich
plasma (2.5 x i07 platelets/100
@l,prepared as described in Ref. 18) were
reagents were of the highest grade available.
Purification of Contortrostatin.
Contortrostatin
incubated with varying concentrations of ‘@I-labeled
contortrostatin for 1 h at
25°C.‘@I-labeled
contortrostatin bound to M24 met cells was separated from
sion assay.
trodon
Two-
contoririx
to 3-week-old
contortrLr
female
venom
scid mice (C.B-17/IcrCrl-scid)
were
was purified from Agkis
by a modification
of our previously
free ‘@I-labeledcontortrostatin
de
phase HPLC (18). The additional final step, cation exchange HPLC, enabled us
to obtain a single isoform of contortrostatin. For this step, contortrostatin was
binding was determined by incubating M24 met cells or platelet-rich plasma
with unlabeled contortrostatin (100-fold molar excess over WI-labeled con
injected into a weak cation exchange HPLC column (carboxymethyl 300;
Synchropak, 250 x 10 mm) purchased from Western Analytical, Inc.
(Temecula,
CA) and equilibrated
with 5 [email protected]
buffer,
by washing twice with 1 ml of ice-cold 2%
BSA in SFM. 1@I-labeledcontortrostatinbound to platelets was separated
from unbound radiolabeled contortrostatin by centrifugation through a layer of
20% sucrose containing 2% BSA as described previously (18). Nonspecific
scribed three-step procedure (18). Briefly, a four-step HPLC procedure was
used, including hydrophobic interaction HPLC and two steps of C18 reverse
tortrostatin at each concentration) for 1 h prior to the addition of tasI@labeled
contortrostatin. Accufit binding software obtained from Beckman was used to
pH 6.0. Elution
was achieved by a 90-mm linear gradient to 1 M sodium chloride in 5 mM
phosphate buffer, pH 6.0. Inhibition of platelet aggregation was used to assay
determine
activity
of M24 met cells stained with anti-integrin antibodies was conducted by
during
purification
as described
previously
(18).
C18 reverse phase HPLC column
(Vydac
218TP54;
Western
Ma
of binding
sites/cell.
fluorescence-activated cell sorter (FA@Star plus, Becton Dickinson).
Inhibition ofBinding ofM24 Met Cells to Immobilized Contortrostatin.
Contortrostatin was immobilized in the wells of 96-well plates at concentra
tions that could support 50—80%of maximum M24 met cell binding. M24 met
trostatin was performed after dissolving the proteins in water-acetonitrile
acetic acid (50/50/0.1) and injecting 5-pJ aliquots of the resulting solution into
to an electrospray
and number
met cell staining. Preimmune IgG was used as control. Cells were sorted on a
lytical, Inc.). PE-contortrostatin was prepared according to the protocol of
Friedmen et a!. (25). ESI mass spectroscopy of contortrostatin and PE-contor
an infusion line connected
constants
of M24 Met Cells. Single color flow cytometric analysis
labeling 2 x i0@ M24 met cells with various dilutions of the test antibody
followed by fluorescein isothiocyanate-labeled goat anti-mouse IgO or goat
anti-rabbit IgG. Several different dilutions of antibodies were tested for M24
ES! Mass Spectroscopy of Contortrostatin.
In preparation for mass spec
troscopy, contortrostatin and reduced PE-contortrostatin were desalted on an
analytical
the affinity
Flow Cytometry
ion source attached to a quadruple
scanning from 400 to 2000 mass/charge (m.z) ratio and scans containing the
cells were treated as described for the cell adhesion assay except that prior to
their addition to the wells they were preincubated with varying concentrations
of GRGDSP peptide, EDTA, anti-integrin antibodies, or preimmune rabbit
ions of interest were extracted
serum IgG for 20 ruin at 25°C. The cell adhesion
mass spectrometer (Sciex API HIR, Toronto, Canada). The same solvent was
pumped through the infusion line at 10 @.tl/min.Data were collected by
from the background.
The data were represented
assay was then performed
as
as mlz ratio and deconvoluted to obtain the mass of the proteins by using the
described above. Maximum percentage of inhibition of M24 met cell adhesion
software
by preimmune antibody was always less than 10%; therefore, nonspecific
inhibition due to anti-integrin antibody was not taken into consideration.
Experimental Metastasis in Mice. M24 met cells (5 X 10@/200 @lof
SFM) were injected into the tail vein of scid mice. After 20 days the animals
myoglobin
provided
with the spectrometer.
The instrument
was calibrated
using
as a standard.
Immobilization of Proteins. Human ECM proteins (fibronectin, vitronec
tin, type I collagen, and laminin) and contortrostatin were suspended in PBS.
In order to establish the optimum concentration of adhesive protein for tumor
cell binding, varying concentrations of each protein were immobilized on the
wells of 96-well microtiter plates overnight at 4°C.Unbound protein was
removed by washing with PBS and excess reactive sites in the wells were
blocked with 2 mg/ml BSA in PBS. Extent of adhesion of M24 met cells was
assayed (as described below) and the lowest concentration of immobilized
protein that could support 50—80% of maximal binding was used for all
were sacrificed and examined for lung colonization.
The lungs were stained
with 15% India ink and fixed in 5% acetic acid, 10% formaldehyde, and 70%
ethanol (27). The number of tumor foci on the surface of the lungs was counted
under a low magnification microscope. To examine the effect of contortrostatin
on lung colonization, 5 X 10@M24 met cells were preincubated with varying
concentrations of the disintegrin for approximately 5 mm in a final volume of
200 @.&l
and injected into the tail vein of acid mice.
subsequent experiments.
RESULTS
Cell Adhesion Assay. To monitor the extent of M24 met cell adhesion, we
used an assay based on the ability of mitochondrial dehydrogenase of viable
Purification of Contortrostatin. We havepreviouslyreportedthe
cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide
into formazan (26). Exponentially growing M24 met cells were harvested with
purification of contortrostatin from Agkistrodon contortrix contortrix
venom (1 g) by a three-step HPLC procedure using hydrophobic
trypsin, washed twice with media containing 10% serum, followed by two
washes with SFM. The cells were suspended at a concentration of 5 X 10@/ml interaction HPLC and two steps on C18 reverse phase HPLC (18).
in SFM. One hundred
@lof cell suspension was added to the microtiter plate
However, for large scale preparation of contortrostatin from crude
wells containing the immobilized protein and the cells were allowed to adhere venom (10 g) we have added an additional step of weak cation
for 1 h at 37°C.Unbound cells were removed by washing with SFM. 3-(4,5- exchange HPLC. This step separated several minor isoforms of con
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
dye was added to each
well and the plate was incubated at 37°C.After 4 h, 100 pi of dimethyl
sulfoxide was added to solubilize the formazan crystals and the plate was
incubated
at room temperature
overnight.
Absorbance
at 570 nm was recorded
using an enzyme-linked immunosorbent assay plate reader. For inhibition of
cell adhesion, varying concentrations of contortrostatin were preincubated with
M24 met cells (5 x 10@/ml)for 20 mm at 25°Cprior to their addition to the
wells.
lodination ofContortrostatin.
Iodogen (Pierce, Rockford, IL) was used to
iodinate contortrostatin according to the manufacturer's protocol. Briefly, 1
mCi of sodium ‘@I-iodide
(Amersham, Arlington Heights, IL) was added to
iodogen-coated
tubes containing contortrostatin
(100 p@g)and iodination was
tortrostatin from the major isoform (data not shown). All isoforms
possessed platelet aggregation-inhibitory activity and migrated to the
same molecular weight in SDS-PAOE (data not shown). The minor
isoforms
from
‘@I-labeled contortrostatin
by gel filtration
using
a PD1O
characterized
due to their low yields.
column (Pharmacia, Piscataway, NJ) equilibrated with PBS. Greater than 93%
conditions (18). The mass of intact, unreduced contortrostatin
of ‘@I-labeled contortrostatin
determined
roacetic
acid. A Beckman
radioactivity
was precipitable
y counter with an efficiency
by 10% trichlo
of 40% was used to
monitor radioactivity. Specific activities of 25—27Ci/mmol were routinely
The
of contortrostatin
indicates that it migrates at approximately
Mr
15,000 under nonreducing conditions and M1 6,000 under reducing
allowed to proceed for 5—6mm at room temperature. Excess iodine was
separated
were not further
major isoform was used for all subsequent characterization. Approx
imately 8—10mg of four-step-purified contortrostatin was obtained
from 10 g of crude venom.
Mass Spectroscopy of Contortrostatin.
In order to assess homo
geneity and obtain an accurate assessment of the molecular mass of
contortrostatin we used ESI-mass spectroscopy. SDS-PAOE analysis
by mass spectroscopy
is 13,505 daltons
as
(data not shown).
The mass of PE-contortrostatin is approximately 8000 daltons (data
not shown). This is the expected value for the individual chains
4994
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CONTORTROSTATININHIBITh MELANOMA ADHESION AND METASTASIS
(approximately 6,750 daltons) of a dimer of 13,505 daltons, taking
into account the incorporation of approximately 1,250 mass units for
12 pyridylethyl groups incorporated into 6 reduced disulfide bonds
(based on homology with other known disintegrins) (12).
Flow
Cytometric
Analysis
of M24
Met
Cells.
100
@80
C
0
The cell surface
expression of several integrin receptors was examined by flow cyto
metric analysis. The mean fluorescent intensities obtained for the
anti-integrin
antibodies
tested
are shown
in Table
1. These
60
.0
40
-C
C
results
20
indicate that M24 met cells express a diverse array of integrins on
0
their surface.
10
100
1000
10000
Inhibition of M24 Met Cell Adhesion to Extracellular Matrix
Contortroatatin
(nil)
Proteins. Adhesive proteins at concentrations that could support 50—
80% of maximal M24 met cell adhesion were used for these experi
Fig. 1. Dose-dependent inhibition of M24 met cell adhesion to extracellular matrix
ments. Optimal binding of M24 met cells to immobilized type I proteins by contortrostatin. M24 met cells (5 X 10@/mi)in the presence of varying
of contortrostatin or SFM were preincubated for 20 mm at 25°Cand
collagen, fibronectin, and vitronectin was achieved using 5 p@g/mlof concentrations
100-s.daliquots were added in triplicate to 96-well plates precoated with 0.5 gs@Jwellof
the ECM proteins (0.5 g.tg/well). Optimal binding to laminin occurred
either type I collagen (A), vitronectin (0]), fibronectin (0), or 1 p@g/wdllof laminin (•)
at 10 @.&Wml.
Contortrostatin
inhibited
adhesion
and the cell adhesion assay was performed as described in “Materials
and Methods.―
of M24 met cells to
Results are expressed as the percentage of inhibition of adhesion observed in the absence
of contortrostatin. The data are representative of three experiments. Points, mean of
fibronectin, vitronectin, and type I collagen in a dose-dependent
manner (Fig. 1). Contortrostatin appeared to be most effective in
inhibiting adhesion of M24 met cells to type I collagen (IC50 = 20
mA), followed
@
by inhibition
of adhesion
to vitronectin
(IC50
triplicate determinations.
SDs were less than 12% of mean values.
= 75 nM)
and to fibronectin (IC50 = 220 nM). Contortrostatin does not signif
icantly inhibit adhesion of M24 met cells to laminin (Fig. 1).
Scatchard Analysis of Contortrostatin
Binding to M24 Met
Cells. We used Scatchard analysis to determine whether contortro
statin binds to M24 met cells in a saturable and displacable manner.
Two different methods were used to determine the number of binding
sites and the affinity of contortrostatin binding to M24 met cells and
platelets. Both displacable (data not shown) and saturable binding
analyses indicate that contortrostatin binds to at least two sites on the
M24 met cells (Fig. 2). More than 90% of 1@I-labeled contortrostatin
binding to M24 met cells can be displaced by 100-fold molar excess
of unlabeled contortrostatin (Fig. 14). Data analysis performed with
Accufit saturation software from Beckman are consistent with a
two-site model. Contortrostatin binds to 165,000 sites/cell with a Kd
of 3 nM and to 500,000 sites/cell with a Kd of 60 nM (Fig. 2B). In
contrast, Scatchard analysis indicated that contortrostatin binds to
only one receptor on human platelets with a Kd of 25 n@tand 100,000
sites/platelet (data not shown).
Inhibition of Binding of M24 Met Cells to Immobilized Con
tortrostatin.
Contortrostatin
immobilized on 96-well microtiter
plates can support binding of M24 met cells in a dose-dependent
manner. At 0.05 p.g/well, contortrostatin supports one-half maximal
binding of M24 met cells (data not shown). Binding of M24 met cells
to immobilized contortrostatin (0.05 @g/well)isblocked by ORODSP
findings indicate that contortrostatin binds to integrin receptors on the
surface of M24 met cells via an ROD-mediated mechanism.
Several anti-integrin antibodies were used to determine the type of
integrin receptors involved in the binding of M24 met cells to con
tortrostatin. The anti-f31 integrin monoclonal antibody mAb 13, which
blocks human lung fibroblast (W138) cell binding to fibronectin (22),
completely inhibits binding of M24 met cells to fibronectin (0.5
@Wwell)and to type I collagen (0.5 @.aWwell).However, it does not
inhibit binding to vitronectin (0.5 @Wwell).Furthermore, mAb 13
inhibits binding of M24 met cells to immobilized contortrostatin (0.05
p@g/well)in a dose-dependent manner (Fig. 4A). These findings mdi
cate that the
integrin receptor is involved in binding M24 met cells
to contortrostatin. The anti-a5(31 integrin antibody, which is specific
for the fibronectin receptor, also effectively blocks M24 met cell
binding to contortrostatin (Fig. 4B). These results indicate that con
(500 @g/ml)
and EDTA (5 mM)(Fig. 3). Since integrin receptors
contortrostatin
require
antibody LM609 (anti-aj33) does not inhibit adhesion of M24 met
cells to immobilized vitronectin (0.5 pg/well) or immobilized
contortrostatin (0.05 p@g/well) (data not shown).
Inhibition of Experimental
Metastasis. To test the biological
activity of contortrostatin in vivo, we used an experimental lung
colonization model using scid mice. Treatment of 5 X 10@M24 met
cells with 20 or 100 ,.@gof contortrostatin prior to injection into the
lateral tail vein of scid mice inhibited the number of tumor foci in the
lungs by 51 and 73%, respectively, as compared to mice who were
given injections of the same number of M24 met cells pretreated with
SFM (Table 2). These data reveal that contortrostatin is a potent
inhibitor of human melanoma cell extravasation in vivo.
metal ions for noncovalent
association
of their subunits,
Table 1Expression of integrin receptors onM24 metcells%
positive―IntegrinAntibody(SD)g3@P4C1O98(1)f33RUU-PL
(5)a@135P3G2
7F1237
(26)a@I33
or P1F652
(25)
97(1)
pAbb
a5@31
(3)a2P1E697(2)a3P1B593(5)a5P1D663(5)ControlIgG1
pAb36
ft,j33/@3LM609
93
these
tortrostatin
inhibits
adhesion
of M24 met cells to type I collagen
and
fibronectin by binding to (3@integrin receptors. Interestingly, despite
the widespread presence of the a2 integrin subunit on M24 met
cells (Table 1), the a2 integrin receptor antibody (P1E6) does not
inhibit adhesion of M24 met cells to type I collagen (0.5 p@g/ml)or
contortrostatin (0.05 p@g/ml)(data not shown).
The anti-vitronectin receptor antibody P302 (anti-aj35) com
pletely inhibits adhesion of M24 met cells to immobilized vitronectin
(0.5 p.g/well)butdoesnotinhibittumorcell adhesionto immobilized
(0.05
,@g/well)
(Fig.
4C).
Anti-vitronectin
receptor
DISCUSSION
@
a
@itive represents
b pAb, polyclonal
C
@5j33/@
polyclonal
the average
of at least
three
determinations.
antibody.
antibody
that
recognizes
both
ct,@3
and
a@5.
I3@ and
3@ subfamiies
of
integrins
play
an
important
role
in tumor
invasion and dissemination (28). Agents that disrupt interactions of f3@
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CONTORTROSTATIN
INHIBIThMELANOMA
ADHESIONAND METASTASIS
Most of the earlier reports of disintegrins
A.
4000
E
a.
0
3000
.u
C
@
focussed
on their inhibi
tion of platelet aggregation (9—18)and their use as antithrombotic
agents (19—21). However, there have been a few publications in
which disintegrins have been reported to inhibit tumor cell adhesion to
ECM proteins. Trigramin has been shown to inhibit human melanoma
C32 cell adhesion to fibronectin (30). Also, albolabrin (31), batrox
ostatin (32), and triflavin (33) inhibit adhesion of murine melanoma
B16F1O cells to ECM proteins including fibronectin, type I collagen,
and laminin. Scatchard analysis of disintegrin binding to tumor cells
has not been reported to date. Identification of integrin receptors and
the affinity of contortrostatin binding to human melanoma cells have
been specifically investigated in the present report.
The finding that immobilized contortrostatin can support adhesion
5000
2000
1000
of M24 met cells
0
50
100
150
200
1251—Contortrostatln
receptors
250
suggests
that this binding
on tumor cells. Adhesion
involves
cell surface
of M24 met cells to immobilized
contortrostatin is completely blocked by GRGDSP peptide and EDTA
(Fig. 3). Therefore, contortrostatin
binds primarily to integrin
receptors on M24 met cells via its ROD site.
Integrin-mediated tumor cell adhesion to vitronectin and fibronec
tin is largely mediated by the ROD sequence present in these proteins
(1—4).Contortrostatin inhibits M24 met cell adhesion to vitronectin
and fibronectin with IC50 values of 75 and 220 nips,respectively (Fig.
1). The binding of type I collagen to integrins was initially believed to
be non-ROD dependent (1). However Cardarelli et a!. (34) have
nil
B.
0.10
0.08
V
V
shown
L_
that a cyclic
ROD
peptide
inhibits
the binding
of type I
collagen to a2(31 in human osteosarcoma cells (MO-63) and human
.u
fibrosarcoma
C
@
0.04
cells (HT1O8O) (34). Furthermore,
triflavin,
an RGD
containing disintegrin (280 nM), inhibits the adhesion of B16F1O cells
to type I collagen
by 89% (33). This suggests
that an ROD recognition
sequence may be involved in the binding of type I collagen. Since the
0.02
a2 integrin
0.00
0
1000
2000
3000
antibody
does not block
M24 met cell adhesion
to type I
collagen or to contortrostatin (data not shown), it appears that these
cells may not use the a2(31 receptor to adhere to type I collagen. There
could be two possible mechanisms to explain why contortrostatin
4000
Bound cpm
effectively
Fig. 2. Scatchard analysis of contortrostatin binding to M24 met cells. M24 met cells
blocks
the adhesion
of M24 met cells to type I collagen
with an IC50 value of 20 ni@i(Fig. 1): (a) the ROD sequence in
(5 X 10@/100@l)
were incubatedat 2.5°C
with varying concentrationsof WI-labeled
contortrostatin
contortrostatin in the absence or presence of 100-fold molar excess of unlabeled contor
quence present in type I collagen; or (b) another epitope besides the
ROD sequence in contortrostatin may be competing for binding of
type I collagen to its receptor. Further elucidation of disintegrin
mediated inhibition of type I collagen binding to M24 met cells should
provide more insight into the mechanisms involved in the binding of
type I collagen to its receptor.
trostatin in a final volume of 250 s.d.WI-labeled contortrostatin bound to tumor cells was
separated from unbound WI-labeled contortrostatin as described in “Materials
and Meth
ods.―
A, saturation analysis of ‘@l-labeled
contortrostatin binding to M24 met cells. D,
total binding; V, nonspecific binding; •,specific binding. B, Scatchard analysis of
saturation binding of ‘@‘l-labeled
contortrostatin to M24 met cells. These results are
representative of three experiments. Points, average of duplicate determinations.
may be displayed
with similar topography
to the se
and/or 133subclass of integrins should have significant antimetastatic
activity. We have previously shown that contortrostatin is a potent
inhibitor of the 13@subclass of integrin receptors, particularly the
tim(Fig.1).Laminin
isknown
tobind
toboth
integrin
andnonintegrin
a1@fi3 (GPIIb/IIIa)
imally inhibits adhesion
receptor
that is involved
in platelet
Contortrostatin
receptors
aggregation
(18). We now report that contortrostatin is also a potent inhibitor of (3@
integrin-mediated adhesion in human metastatic melanoma cells.
Contortrostatin appears to be unique among the disintegrins re
ported to date. Disintegrins usually exist as monomers with molecular
masses ranging from 5 to 9 kilodaltons (9—17). Chao et a!. (12)
originally reported that applaggin, the disintegrin from Agkistrodon
piscivorus piscivorus venom, was a dimer. However, a more recent
report using mass spectroscopy reveals that applaggin exists as a
monomer with a mass of 7666 daltons (29). Thus, contortrostatin with
a molecular mass of 13.5 kilodaltons appears to be the largest disin
tegrin. Furthermore, contortrostatin appears to exist as a dimer since
mass spectrometry of reduced PE-contortrostatin indicates a mass of
approximately 6750 daltons for the individual subunits. SDS-PAGE
analysis
indicates
that reduced
and nonreduced
contortrostatin
migrate
at approximately 6 and 15 kilodaltons, respectively (18), which
provides further support that contortrostatin is a dimer.
has minimal
(35), which
could
100
effect on tumor cell adhesion
explain
why contortrostatin
to lami
only mm
of M24 met cells to laminin (Fig. 1).
-F--
80
C
0
60
‘I
0
.C
V
.(
40
20
r@
0
C
GRGDSP
ii
EDTA
Fig.3. Inhibitionof adhesionof M24metcellsto immobilizedcontortrostatin.M24
met cells (5 x 105/mi)were preincubated with serum-free media (C), GRGDSP peptide
(0.5 mg/mI), or EDTA (5 mM) for 20 miii at 25°Cand 100-sal aliquots were added to
96-well plates coated with 0.05 @gJwell
of contortrostatin. The assay was performed as
describedin “Materials
andMethods.―
Columns,meanof six determinations;
bars, SD.
4996
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1994 American Association for Cancer Research.
CONTORTROSTATIN INHIBITh MELANOMA ADHESION AND METASTASIS
integrin. Additionally, mAb 13 inhibits adhesion of M24 met cells to
fibronectin and contortrostatin (Fig. 4A). Contortrostatin appears to
bind specifically to the fibronectin receptor a5(31 because an anti-a5(31
integrin antibody also inhibits binding of M24 met cells to contortro
statin (Fig. 4B). Furthermore, contortrostatin completely blocks ad
hesion of M24 met cells to immobilized fibronectin with an IC50 of
220 flM (Fig. 1). Our findings therefore demonstrate that contortro
statin binds to (3@integrin receptors on the surface of M24 met cells.
Binding of contortrostatin to the (3@integrin subfamily (e.g., the a5135
receptor) most likely accounts for at least one of the binding sites
indicated by the biphasic Scatchard plot (Fig. 2B). However, it is not
known at this time whether both species of binding sites involve the
B.
A.
100
@80
@
0
..@
60
40
C
—
20
0
10
100
Anti—ftl (Mg/mI)
0
20
40
60
AntI—a5@1 (MI/mI)
I3@ integrin
@
@
receptors
or
if contortrostatin
binds
to
other
members
of
the (3 integrin subfamily (e.g., the f3@and/or
subfamilies).
C.
Contortrostatin is a potent inhibitor of adhesion of M24 met cells to
1 00
immobilized vitronectin with an IC50 of 75 nM (Fig. 1). Melanoma
cells bind to vitronectin via the aj3@ (3, 5, 7, 24), a@3@(3, 5, 7, 24),
@80
and/or a@f3i(7) integrin receptors. Flow cytometric analysis indicates
C
60
0
that 36% of M24 met cells are a,,j3@positive (Table 1). These results
.@
40
are also supported by Mueller et a!. (22). We found that the anti-cçj33
.C
C
monoclonal antibody (LM609) did not inhibit adhesion of M24 met
— 20
cells to immobilized vitronectin or to immobilized contortrostatin
0
(data not shown). This finding suggests that M24 met cells do not use
5
10 15 20 25
0
the
receptor to bind to vitronectin. Furthermore, the anti-(31
Anti—av@5 (MI/mi)
monoclonal antibody (mAb 13) did not inhibit adhesion of M24 met
cells to immobilized vitronectin, thereby suggesting that these cells
Fig. 4. Antibody-mediated inhibition of adhesion of M24 met cells to immobilized
proteins.A,M24metcellswerepreincubatedwithanti-a, (mAb13)antibodyfor20ruin probably do not utilize the a@,f31receptor to bind vitronectin (Fig. 4A).
at 25°Cand 100-Ml aliquots were added to wells coated with fibronectin (0), type I
Greater than 50% of M24 met cells stain positive for cçj3@
surface
collagen (A), contortrostatin (•),or vitronectin (0). B, M24 met cells were preincubated
with anti-fibronectin receptor (ct5@1)antibody for 20 mm at 25°Cand 100 Ml was added
expression (Table 1). Furthermore, anti-cçj35 (P302) completely
to wells coated with 0.5 pg/well of fibronectin (0) and 0.05 pg/well of contortrostatin
blocks adhesion of M24 met cells to immobilized vitronectin, sug
(•).
C,M24 metcells
werepreincubated
withanti-vitronectin
receptor
(cr.@)
antibody
gesting that a@135may be the primary receptor involved in adhesion of
P3G2 for 20 ruin at 25°Cand 100 p.1was added to wells coated with 0.5 pg/well of
vitronectin @J@J)
and 0.05 pg/well of contortrostatin (•).The cell adhesion assay was
M24 met cells to vitronectin (Fig. 4C). However, P302 did not inhibit
performed as described in “Materials
and Methods.―
Points, mean of six determinations;
adhesion
of M24 met cells to immobilized contortrostatin (Fig. 4C).
bars, SD.
One possible interpretation of this finding is that the vitronectin
receptor epitopes recognized by P302 may differ from the epitopes
Nonetheless, our results conclusively demonstrate that contortro
recognized by contortrostatin. Alternatively, contortrostatin may have
statin is an effective and selective inhibitor of integrin-mediated
a much higher affinity for M24 met cells (Fig. 2) compared to P3G2.
adhesion of M24 met cells to ECM proteins.
Therefore immobilized contortrostatin may displace P302 bound to
This report is the first to our knowledge that demonstrates disinte
M24 met cells in the cell adhesion assay (Fig. 4C).
grin binding to two sites on tumor cells. This binding characteristic of
Contortrostatin is an effective inhibitor of lung colonization of
contortrostatin could be attributed to its dimeric structure. All disin
human metastatic melanoma cells (Table 2). It is a much more potent
tegrins reported thus far have a single binding site on platelets with Kd inhibitor of experimental metastasis in mice than an ROD-containing
values ranging from 10 to 120 nt@i(9—17). Scatchard analysis of
synthetic peptide (ORODS). Humphries et a!. (36) reported that the
contortrostatin binding indicates that it binds to two sites on M24 met
ORODS peptide at 1.5 and 3 mg when coinjected with 7 X i04
cells (Fig. 4) but to only a single site on platelets. Contortrostatin has
B16F1O cells inhibited lung colonization in C57BL/6 mice by 50 and
a higher affinity toward M24 met cells (Kd = 3 nM) than to platelets
90%, respectively (36). In contrast, contortrostatin at only 20 and 100
(Kd
25 flM).
I.Lg inhibits
lung colonization
of 5 X 10@ M24 met cells by approxi
Since contortrostatin binds to integrins on M24 met cells, we
mately 50 and 75% in scid mice, respectively, (Table 2). Determining
designed experiments to identify the specific integrin receptors in
the effect of the ROD peptide on murine melanoma cells in C57BL/6
volved in binding. Flow cytometric analysis indicates that the I3@
subfamily of integrins are major receptors on the these cells (Table 1).
Anti-(31
(mAb
13) antibody
was used because
type I collagen
contortrostatinM24
Table 2 Inhibition of experimental metastasis by
and
fibronectin bind to the f3@subfamily of integrins (1, 2). mAb 13
inhibits adhesion of M24 met cells to type I collagen (Fig. 4A) but an
antibody to the a2 integrin subunit (P1E6) does not appear to inhibit
M24 met cell adhesion to type I collagen or to contortrostatin. Al
though flow cytometric analysis indicates that more than 90% of M24
met cells are positive for a2 (Table 1), it appears a (3@integrin receptor
different from a2f31 (e.g., a1(31 and/or a3(31) may be the major
receptor on these cells for type I collagen. Interestingly, M24 met cells
are 93% positive for surface expression of the a3 integrin subunit
(Table 1). Due to the fact that the a1 integrin antibody was unavailable
to us, the cells were not analyzed for surface expression of this
(20and met cells (5 X 10@)were treated in the absence or presence of contortrostatin
lateraltail
100 @a)in serum-free media in a final volume of 200 @dand injected into the
stainedwith
veinsof scidmice.After20 daysthe animalsweresacrificedandlungswere
lowpower
Indiaink.Tumorcolonieson the surfaceof the lungswerecountedundera
dissecting microscope.Average
ofCN―
no.
No.of
@.tg/mouse
values@'0 animals
020
<0.0001100
lungcolonies'
Inhibition
animal (SEM)
(%)
10
9
405(20)
199(19)
51
10
111(16)
73
aCN,
contortrostatin.
b ,@values for control versus 20 and 100
20 versus 100 @.tg
of contortrostatin,
P
<0.0001
@g
of contortrostatin, respectively. P value for
<0.005.
4997
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1994 American Association for Cancer Research.
CONTORTROSTATIN
IN}tlBfl'5
MELANOMA
mice versus the effect of contortrostatin on human melanoma cells in
scid mice may not be an appropriate comparison. Nonetheless, on a
molar basis contortrostatin appears to be at least 2000-fold more
potent than the RGD peptide.
This is the first report to our knowledge that demonstrates efficacy
of a disintegrin
as an inhibitor of extravasation
contortrostatin
possesses
METASTASIS
Echistatin: a potent platelet aggregation inhibitor from the venom of the viper, Echis
L A., and Maraganore,J. M. Agkisrrodonpiscivoruspiscivorusplateletaggregation
inhibitor: a potent inhibitor of platelet activation. Proc. NatI. Acad. Sci. USA, 86:
8050—8054,1989.
13. Williams, J., Rucinski, B., Holt, J., and Niewiarowski, S. Elegantin and albolabrin
purified peptides from viper venoms: homologies with the RODS domain of fibrin
several unique features that
distinguish it from all reported disintegrins. It has a large molecular
mass and a dimeric structure. Contortrostatin is a potent inhibitor of
human metastatic melanoma cell adhesion to type I collagen, vitronec
ogen and von Wilebrand factor. Biochim. Biophys. Acts, 1039: 81—89,1990.
14. Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell,
A. M., Nannizzi, L, and Charo, I. F. Barbourin.A GPIIb-IIIaspecific integrin
antagonist from the venom ofSistrurus m. barbouri. J. Biol. Chem., 266: 9359-9362,
1991.
15. Huang, T. F., Sheu, J. R., Teng, C. M., Chen, S. W., and Liu, C. S. Triflavin, an
antiplatelet Arg—Gly—Asp-containing
peptide, is a specific antagonist of platelet
membrane glycoprotein llb-IlIa complex. J. Biochem., 109: 328—334,1991.
16. Gould. R. J., Polokoff, M. A., Friedman, P. A., Huang, T. F., Holt, J. C., Cook, J. J.,
and Niewiarowski, S. Disintegrins: a family of integrin inhibitory proteins from viper
venoms. Proc. Soc. Exp. Biol. Med., 195: 168—171,1990.
17. Williams, J. A. Disintegrins: ROD-containing proteins which inhibit cell/matrix
interactions (adhesion) and cell/cell interactions (aggregation) via the integrin recep
tors. Pathol. Biol., 40: 813—821,1992.
18. Trikha, M., Rote, W. E., Manely, P. J., Lucchesi, B. R., and Markiand, F. S.
Purification and characterization of platelet aggregation inhibitors from snake yen
oms. Thromb. Res., 73: 39—52,1994.
19. Yasuda, T., Gold, H. K., Leinbach, R. C., Yaoita, H., Fallon, J. T., Guerrero, L,
Napier, M. A., Bunting, S., and CoHen, D. Kistrin, a polypeptide platelet GPIIbIIIIa
receptor antagonist, enhances and sustains coronary arterial thrombolysis with re
combinant tissue-type plasminogen activator in a canine preparation. Circulation, 83:
1038—1047,1991.
20. Holahan,M. A., Melloft,M. J., Oarsky,V. M., and Shebuski,R. J. Preventionof
reocclusion following tissue type plasminogen activator-induced thrombolysis by the
tin, and fibronectin. It is the only disintegrin reported thus far that
@
AND
carinatus. J. Biol. Chem., 263: 19827—19832,1988.
12. Chao, B. H., Jakubowski, J. A., Savage, B., Chow, E. P., Marzec, U. M., Barker,
of human melanoma
cells. However, albolabrin (31) and triflavin (33) at similar concen
trations to contortrostatin almost completely inhibit lung colonization
of murine melanoma cells (B16F1O, 1 X 10@) in C57BL,/6 mice.
Interestingly, albolabrin inhibits murine melanoma cell adhesion to
laminin (31), whereas contortrostatin has a minimal effect on adhesion
of human melanoma cells to laminin (Fig. 1). By contrast, contortro
statin inhibits melanoma cell binding to type I collagen and vitronec
tin (Fig. 1), which was not reported for albolabrin. Disintegrin
mediated inhibition of lung colonization could involve interference
not only with tumor cell-lung matrix interactions but also tumor
cell-platelet interactions and tumor cell-vascular endothelial cell in
teractions. Mechanisms involved in the antimetastatic activity of
contortrostatin are presently being investigated in our laboratory.
In conclusion,
ADHESION
ROD-containingpeptide,echistatin,in a canine modelof coronarythrombosis.
Pharmacology, 42: 340—348, 1991.
binds to two sites on tumor cells, while binding to a single site on
21. Shebuski, R. J., Stabiito, I. J., Sitko, 0. R., and Polokoff, M. H. Acceleration of
platelets. We have previously shown that contortrostatin is a
recombinant tissue-type plasminogen activator-induced thrombolysis and prevention
integrin antagonist (18) and we now report that it is also a f3@
of reocclusion by the combination of heparin and the Arg—Oly—Asp.containing
peptide bitistatin in a canine modelofcoronary thrombosis. Circulation, 82: 169-177,
integrin antagonist. Finally, contortrostatin is a potent inhibitor of
1990.
lung colonization of human metastatic melanoma cells in vivo.
22. Mueller,B. M.,Romerdahl,C. A.,Trent,J. M.,and Reisfeld,R. A. Suppressionof
spontaneous melanoma metastasis in scid mice with an antibody to the epidermal
growth factor receptor. Cancer Res., 51: 2193—2198,
1991.
ACKNOWLEDGMENTS
23. Aldyama,S. K., Yamada,S. S., Chen,W. T., and Yamada,K. M. Analysisof
fibronectin receptor function with monoclonal antibodies: roles in cell adhesion,
migration, matrix assembly, and cytoskeletal organization. J. Cell Biol., 109:
We acknowledge Dr. Kym Faull and Ken Konklin (University of California,
Los Angeles) for mass spectrometric analysis of contortrostatin. We also
acknowledge Dr. Ralph Reisfeld (The Scripps Research Institute, La Jolla, CA)
for providing the human metastatic melanoma (M24 met) cells used in this
study. We thank Drs. Zoltan Tokes and especially Jon Backstrom for their
863—875,
1989.
24. Wayner, E. A., Orlando, R. A., and Cheresh, D. A. Integrins av@3 and av@5
contribute to cell attachment to vitronectin but differentially distribute on the cell
surface. J. Cell BioL, 113: 919—929,1991.
25. Friedman,M.,Krull,L H.,andCavins,J. F. Thechromatographic
determinationof
cystine and cysteine residues in proteins as S-@-(4-pyridylethyl)cysteine.J. Biol.
advice throughout the period of this study.
Chem.,245:3868—3871,
1970.
26. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays. J. ImmunoL Methods, 65: 55—63,1983.
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Contortrostatin, a Snake Venom Disintegrin, Inhibits β1
Integrin-mediated Human Metastatic Melanoma Cell Adhesion
and Blocks Experimental Metastasis
Mohit Trikha, Yves A. De Clerck and Francis S. Markland
Cancer Res 1994;54:4993-4998.
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