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[CANCER RESEARCH 45, 4048-4052, September 1985]
Inhibitory Effects of Dipyridamole on Growth, Nucleoside
Incorporation, andPlatelet-activating Capability in the U87MG and
SKNMC Human Tumor CellLines1
Eva Bastida,2 Josefina del Prado, Lourdes Almirall, G. A.
Jamieson, and Antonio Ordinas
Hospital ClÃ-nico,Servicio de Hemoterapia y Hemostasia,
Universidad de Barcelona, Casanovas, 143, 08036 Barcelona, Spain
[E.B., J.d.P., LA., A.O.], and American RedCross
BiomédicalResearch Laboratories, Bethesda, Maryland [E.B., G.A.J.,
A.O.]
ABSTRACT
The effects of dipyridamole on tumor cell function were examined
in cultures of two lines of human origin, the SKNMCneuroblastoma
line that activates platelets by a mechanismwhich is dependent on
the release of adenosine 5'-diphosphate
and the U87MG glioblastoma line that induces platelet
activationby the generation of thrombin. Cells grown in the
presence ofdipyridamole at 1 MM showed >80% inhibition of uptake
ofadenosine, thymidine, and undine with both lines. At 5 nM
tumorcell growth was inhibited by 70% (U87MG) and 90% (SKNMC)but
without concomitant cytotoxicity as determined by clono-genic assay
(50% inhibitory concentration -20 MM). At 10 MMdipyridamole cyclic
adenosine 3':5'-monophosphate levels in
creased 150% with both cell lines but no changes above
baselinevalues were seen at 2.5 MM.The two cell lines showed
differentresponses to being cultured in the presence of
dipyridamole interms of their ability to subsequently activate
platelets. U87MGcells cultured in 10 MM dipyridamole showed a
doubling of thelag time as compared with cells grown in the absence
of dipyridamole but with full aggregation; with SKNMC cells the
aggregation rate was reduced and cells grown in 10
MMdipyridamoleshowed no reversible first wave, a 5-fold increase in
lag time and
a 75% inhibition in total aggregation. Since therapeutic doses
ofdipyridamole result in plasma concentrations of ~3.5 MM these
results suggest that potential antimetastatic effects of
dipyridamole could be direct arising from inhibition of important
steps intumor cell metabolism or indirect by suppressing one or
more ofthe mechanisms involved in the ability of tumor cells to
activateplatelets.
INTRODUCTION
Platelets may be involved in the metastatic spread of tumors(7,
26, 30), and although some laboratory findings support
thishypothesis (16, 23) other results are ambiguous (12, 17).
However, recent results showing a reduction in the number of
lungcolonies in animals treated with an antiplatelet antibody
(24)confirm earlier observations about the role of platelets in
thedevelopment of secondary métastases(8).
Several laboratories have found that both animal (9, 11, 14)and
human tumor cells (1, 13, 22) are able to activate plateletsin
vitro. In our laboratory we have characterized two
differentmechanisms of platelet activation by tumor cell lines (2).
Onemechanism (ADP dependent) involves the stimulation of
platelets
'This work was partially supported by the Spanish Comisión
Asesora de
Investigación CientÃ-ficay Técnicano. 962 and by a grant from
Boerhinger IngelheimSpain. Additional support was provided by USPHS
Grants CA 30538 and RR05737.
•To whom requests for reprints should be addressed.
Received 2/7/85; revised 5/28/85; accepted 6/11/85.
by ADP while the other mechanism (thrombin dependent) depends on
the generation of thrombin by tissue factor present onmicrovesicles
shed by the tumor cells (3).
These experimental data suggest that the administration
ofantiplatelet drugs could be of help in the treatment of
cancerpatients. Although some few clinical studies in this respect
havebeen completed they have been inconclusive (10, 20).
Dipyridamole is one of the most used inhibitors of platelet
function (21).Besides its antiplatelet phosphodiesterase activity
(4) dipyridamole has also been shown to increase the synthesis of
vascularprostacyclin (28) and to be a potent inhibitor of
nucleosidetransport (19, 25). Dipyridamole also causes inhibition
of lympho-
proliferation (6), killing of rat hepatoma cells (31), and
enhancement of the toxicity of deoxyadenosine and deoxycoformycin
toa mouse leukemia line (15).
In the present studies we report the effects of dipyridamoleon
the U87MG and SKNMC human tumor cell lines with regardto tumor cell
proliferation, nucleoside incorporation, cAMP3 lev
els, and modifications in tumor cell proaggregant and
procoag-
ulant activities.
MATERIALS AND METHODS
Cell Culture. Two human tumor cell lines were used in the
presentstudy, the U87MG line derived from a glioblastoma and the
SKNMC linederived from a neuroblastoma. Both had been kindly
provided by thelate Dr. JörgenFogh of the Sloan Kettering
Institute, Rye, NY.
U87MG and SKNMC cells were grown in 95% air-5% CO2 as mono-
layers in polycarbonate flasks (Nunc Corp., Denmark) using
minimalessential medium supplemented with 1% nonessential amino
acids,gentamycin (50) mg/ml, 2 mw glutamine, and 20 mw
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer. The
concentration of fetal calf
serum was 15 and 10% for the U87MG and SKNMC cell lines,
respectively. The cells were passaged twice weekly.
For the metabolic studies the tumor cells were harvested by
trypsin-
ization (0.25%) for 2 min and the viability was determined by
trypanexclusion; in no case was viability less than 90%. For
platelet aggregationexperiments and for the procoagulant activity
tests the cells were harvested in the absence of proteases by
decanting the culture medium andthen washing the monolayers twice
with Hanks' balanced salt solution
without Ca2+ and Mg2*. Washed cell monolayers were treated for 1
hwith 5 HIM ethyleneglycol bisOJ-aminoethyl
ether)-/v,/v,/v',/v'-tetraaceticacid in HBSS at 37°C.The detached
cells were then centrifuged (100 x
g for 10 min) and the cell pellets were resuspended in the same
solutionto yield a cell concentration of approximately 1 x 107
cells/ml. Viability
was measured by trypan blue exclusion and in no case was lower
than90%.
Inhibition of Incorporation of Labeled Nucleosides. The
3H-labeled
nucleosides studied here were thymidine (20 iiC\/^mo\),
adenosine (17.8
3The abbreviations used are: cAMP, cyclic AMP; HBSS, Hank's
balanced salt
solution; TCA, trichtoroacetic acid.
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EFFECTS OF DIPYRIDAMOLE ON CULTURED TUMOR CELLS
l), and uridine (27.1 /jCi/itmol) (New England Nuclear,
Boston,MA), made up as solutions of 33 //Ci/ml before use. For
measuring theincorporation of labeled nucleosides, 3 x 105 tumor
cells were seeded
into 2.5 cm macrotiter wells (Costar, Cambridge, MA) containing
2.5 mlof culture medium. Aliquots of 0.5 ml of medium without or
with appropriate concentrations of dipyridamole were immediately
added to thewells. For each experiment triplicate wells were run
for controls and fordipyridamole-treated cells. After 24 h of
incubation at 37°Cthe labeled
nucleosides were added to the culture medium in appropriate
volumesto give a final concentration of 1 /iCi/ml. Incubation was
then continuedfor 1 h at 37°C.After this period of time the medium
was removed, the
wells were maintained on ice, and the cell monolayers were
washedtwice with cold HBSS and three times with 3 ml of cold
trichloroaceticacid (5% w/v) allowing each aliquot of TCA to stand
for 10 min in contactwith the cell monolayers. Finally and at room
temperature the monolayerswere washed twice with absolute ethanol
(10 min each wash). Anyremaining liquid was evaporated under
nitrogen and 1 ml of 0.3 N NaOHwas added to dissolve the residue.
After 1 h at 37°C 750 n\ of the
solution were pipeted from each well, neutralized with 150 n\ of
1.5 NMCI, and placed in a scintillation vial to which 9 ml of
scintillation fluid(Aquasol II; New England Nuclear, Boston, MA)
were added.
Growth Inhibition Studies. Logarithmically growing U87MG
andSKNMC tumor cells were harvested and seeded into 25 cm2 flasks
atconcentrations of 2 x 10°cells/flask. Triplicate samples were
used for
each experiment. In certain experiments a fixed concentration of
dipyridamole was added to the medium and the cells were incubated
fordifferent periods of time. In other experiments graded
concentrations ofthe drug were added to the medium and the cells
were harvested after24 h of incubation. In either case the cells
were harvested by trypsin-
ization (0.25%) for 2 min and the viability was determined by
the trypanblue exclusion test.
Clonogenic Assay. Clonogenic assays were performed as
describedby Salmon ef al. (27). Briefly logarithmically growing
U87MG and SKNMCcells were seeded at 500 cells/25 cm2 flask.
Immediately after this
dipyridamole in various concentrations from 2 to 30 UM was added
andthe cells were allowed to grow. After 7 days incubation the
medium wasdiscarded and the cells were stained with a saturated
solution of crystalviolet. Colonies consisting of more than 50
cells were counted and thenumber of surviving colonies in the
dipyridamole-treated group was
calculated as a percentage of the number of colonies found with
untreated cells. The 50% inhibitory concentration values for the
cytotoxicityof dipyridamole were calculated by linear regression
analysis.
Intracellular cAMP Levels. U87MG and SKNMC cultures grown inthe
presence and absence of dipyridamole were trypsinized after 24 hof
incubation. An aliquot was withdrawn from the cell suspension in
orderto determine the cell recovery and the remainder was
centrifuged. Cellpellets were resuspended in HBSS and sonicated in
a Branson sonifierequipped with a microtip (Branson Sonic Power
Co., Danbury, CT). Thesonicate was treated with 2 ml of cold 5% TCA
and the protein precipitatewas removed by centrifugation at 10,000
x g for 10 min. The supernatantfractions were extracted 5 times
each with 3 volumes of diethyl ether toremove TCA and the extracted
aqueous samples were then evaporatedunder nitrogen at 50°C. The
residues were redissolved in 0.2 ml of
acetate buffer (pH 5.4) prior to assay. cAMP content of the
tumor cellswas determined by radioimmunoassay according to a
standard technique(29) using a commercially available kit (Amersham
Corporation, ArlingtonHeights, IL). Recovery of cAMP was 92 ±10%
(SD) as determined byusing added [3H]cAMP (New England Nuclear,
Boston, MA) and calcu
lated concentrations were corrected accordingly.Platelet
Aggregation. Blood from healthy donors who had not taken
platelet-affecting drugs for at least 10 days before phlebotomy
was used.
Blood was drawn by venipuncture and anticoagulated with heoarin
(5 U/ml). Platelet-rich plasma was prepared by centrifugation at
100 x g for
10 min and platelet aggregation was measured by aggregometry
(22).Clotting Assays. The recalcification time was measured in a
Fibro-
meter (Becton Dickinson, Rutherford, NY). The procoagulant
activity of
the tumor cells was measured using the one-stage assay.
Pooled,citrated, platelet-poor normal human plasma (100 »I)was
incubated with100 /¿Iof the tumor cell suspension at 37°C in the
control buffer for 1
min followed by addition of 100 n\ of 25 mM Cad? and the
clotting timewas recorded.
RESULTS
Effect of Dipyridamole on Tumor Cell Growth. Two differenttypes
of experiments were performed in order to evaluate theeffects of
dipyridamole on tumor cell growth. In one case thenumber of cells
was determined in U87MG and SKNMC culturesduring growth in the
presence of a fixed amount (5 »M)ofdipyridamole (Chart 1). The
growth of both cell lines was reducedcompared with the control
cultures and the SKNMC line wasmore sensitive than U87MG to the
presence of dipyridamole atall of the time points examined.
In a second type of experiment a dose-response curve was
constructed to evaluate the effect of dipyridamole on tumor
cellproliferation (Chart 2). Tumor cells (1 x 105/rnl) without and
with
different concentrations of dipyridamole were seeded into 25cm2
flasks and allowed to incubate at 37°Cfor 24 h. At the end
of this period cultures were harvested and the cell count and
cellviability were determined. Dipyridamole inhibited cell
proliferationin both tumor cell lines in a dose-dependent fashion
and maxi
mum inhibition was reached at approximately 5 UM. AgainSKNMC
cells were more sensitive to dipyridamole than wereU87MG cells at
all concentrations examined. None of the concentrations studied
displayed a cytotoxic effect since the viabilityof the cells
cultured in the presence of dipyridamole was in thesame range as
that of controls (approximately 90%) as measuredby trypan blue
exclusion.
Cytotoxic Effects of Dipyridamole on U87MG and SKNMCTumor Cell
Cultures. Determination of cytotoxicity by dono-
genie assay indicated that the 50% inhibitory concentration
fordipyridamole was 23 UMfor U87MG cells and 19 MMfor SKNMCcells
(Chart 3). These cytotoxic concentrations are much higherthan the
drug concentrations which gave maximal inhibition ofcell growth in
both lines (~5 /¿M).
24 48
TIME OF HARVEST (hours)
72
Chart 1. Effects of 5 UMdipyridamoleon growth of
U87MGcells(•)and SKNMCcells (O) at different harvest times.
Points, means of triplicate experiments; bars,SE.
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EFFECTS OF DIPYRIDAMOLE ON CULTURED TUMOR CELLS
1 103 A 5 6 7 8jjM DIPYRIDAMOLE
Chart2. Effect of dipyridamote concentration on growth of U87MG
(•)andSKNMC (O) cells. Points, means of triplicate assays; bars,
SE.
15 20 25 30
DIPYRIDAMOLE
for [3H]thymidine and [3H]uridine at 0.1 MM(Table 1).
Inhibition
of nucleoside incorporation into SKNMC cells required
higherconcentrations of the drug. Dipyridamole at 1 MMinhibited
incorporation of the three nucleosides tested approximately 80%.
Ata concentration of 10 /¿Mthere was complete inhibition of
nucleoside incorporation with both cell lines.
Effect of Dipyridamole on Intracellular cAMP Levels. Baseline
values for cAMP in cells grown in the absence of dipyridamolewere
3.8 ±0.6 pmols/106 cells and 4.6 ±0.3 pmols/106 cells
for U87MG and SKNMC cells, respectively (Table 2). Culturingthe
cells in the presence of 2.5 MM dipyridamole caused nosignificant
increase in these values. However, at 10 UM dipyridamole
concentrations cAMP values were increased about 150%to a value of
6.2 ±1.1 for U87MG and 8.2 ±2.1 for SKNMC;values were significant
at the level, P
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EFFECTS OF DIPYRIDAMOLE ON CULTURED TUMOR CELLS
CONTROL
10MIN.
Chart 4. Representative tracings of the platelet-aggregating
activity of tumorcells cultured in presence of 2.5 and 10 \a*
dipyridamole.A, platelet-aggregationtracings obtained with 1x10°
U87MG cells/ml; B, platelet-aggregationtracingsobtained with 1 x
10* SKNMC cells/ml.
//Mwere unable to induce the first wave of reversible
aggregationand showed significantly less maximal aggregation (Chart
48).
Procoagulant Activity of Tumor Cells Cultured in the Presence of
Dipyridamole. We have already demonstrated thatUS7MG tumor cells
(but not SKNMC cells) display a strongprocoagulant activity that is
able to significantly shorten therecalcification time of pooled
normal platelet-poor plasma (3). Inthe present work the one-stage
recalcification time was reduced
from 210 ±12 to 60 ±4 s by U87MG cells and this value
wasunaffected when U87MG cells grown in the presence of
dipyridamole were used.
DISCUSSION
Support for the use of antiplatelet drugs in the treatment
ofcancer derives from experimental observations which ascribe arole
to platelets in the spread of malignant disease (8, 24).However,
the direct effect that inhibitors of platelet function mightexert
on tumor cell biology has not been considered.
In our study we have correlated the effects of dipyridamole
onthe growth, metabolism, and platelet-activating activity of
two
tumor cell lines of human origin. Our data clearly show
thatdipyridamole has an inhibitory effect on the growth of both
tumorcell lines studied, that this effect is time and
concentrationdependent, and that concentrations with maximal
inhibitory effectshow no concomitant cytotoxicity. The plasma
levels of dipyridamole when given at therapeutic doses as an
antiplatelet drugreach a maximum value of approximately 3.5 ^M
(21). Thisconcentration is in the same range as the concentrations
we
have found to inhibit the growth of U87MG and SKNMC cells
(5MM)and far below the cytotoxic concentrations determined
byclonogenic assay (~20 /¿M).The results suggest that a
potential
effect of the drug could be directed to the inhibition of tumor
cellgrowth.
Incorporation of nucleoside into tumor cells was also
markedlyinhibited by dipyridamole at concentrations well below
therapeutic plasma levels. Nucleoside transport is the initiating
step in theutilization of exogenous nucleosides for the salvage
pathwayand it is known that the activities of the nucleoside
salvageenzymes are increased in rat and human hepatomas (5,18).
Ourresults demonstrate that dipyridamole at concentrations below1
MM is a powerful inhibitor of nucleoside incorporation in bothtumor
cell lines studied.
The observed increase of intracellular cAMP in the tumor
cellsinduced by dipyridamole could affect their ability to cause
plateletaggregation. It is interesting to note that the platelet
aggregatingability of the U87MG line which activates platelets by a
thrombin-dependent mechanism is less affected when the tumor cells
arecultured in dipyridamole than is the SKNMC line that
operatesthrough an ADP-related mechanism. It is important to point
out
that the procoagulant activity of the U87MG tumor cells
wasunaffected after culturing the cells in the presence of
dipyridamole. The procoagulant activity displayed by this cell line
iscaused by the presence of tissue factor in the tumor cell
membrane (3). Therefore this lack of effect on the generation of
theprocoagulant activity suggests that dipyridamole does not
alterthe structure of the U87MG cell membrane with regard to
theexposure of tissue factor.
The above results suggest that the effect of dipyridamole
ontumor cell metastasis could be direct by inhibiting
importantsteps in tumor cell metabolism such as nucleoside
incorporationor could be indirect by suppressing one or more of the
mechanisms involved in the capability of tumor cells to activate
platelets.
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1985;45:4048-4052. Cancer Res Eva Bastida, Josefina del Prado,
Lourdes Almirall, et al. and SKNMC Human Tumor Cell
LinesIncorporation, and Platelet-activating Capability in the U87MG
Inhibitory Effects of Dipyridamole on Growth, Nucleoside
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