Download Effect of Alkyl-lysophospholipid on Glioblastoma

(CANCER RESEARCH 51, 1713-1719. March 15. 1991]
Effect of Alkyl-lysophospholipid
Brain Tissue in Vitro '
on Glioblastoma Cell Invasion into Fetal Rat
Olav Engebraaten,2 Rolf Bjerkvig, and Michael E. Berens1
The (jade Institute, Department of Pathology, University of Bergen, A'-5021 Bergen, Norway ¡O.E., R. B.f, and The Brain Tumor Research Center, University of
California, San Francisco, San Francisco, California ¡M.E. B.J
ABSTRACT
The antitumor effect of alkyl-lysophospholipid (ALP) was studied on
a continuous glioma cell line (GaMg) as »ellas on tumor spheroids
obtained from three different primary brain tumor biopsies. GaMg monolayer growth was reduced by 50% after treatment with 30 MMALP; cells
accumulated in the G2M phase of the cell cycle as determined by flowcytometric analyses. Tumor spheroid growth was reduced by 25 and 44%
during treatment with 10 and 30 MMALP, respectively. These drug
concentrations also caused a severe destruction of spheroids. No effect
on growth or morphology was seen in spheroids treated with 0.1 and 1.0
MMALP.
ALP caused a dose-dependent inhibition of invasion by GaMg tumor
spheroids into brain aggregates. After 168 h of 1.0 MMALP treatment,
the volume of the intact brain aggregate was 90% larger than that in the
untreated co-cultures.
To further investigate the efficacy of ALP as an anti-invasive drug,
co-cultures were performed with specimens obtained from three primary
brain tumors: a highly invasive glioblastoma multiforme, an anaplastic
astrocytoma, and an astrocytoma. Treatment of spheroids from the most
invasive tumor with ALP caused a 7-fold preservation of normal brain
tissue relative to control co-cultures. Moreover, the sensitivity of primary
glioma spheroids to the anti-invasive effect of ALP seemed to be associ
ated with the aggressiveness of the tumor; spheroids from the more
malignant specimen (glioblastoma multiforme) were more sensitive than
those from the less aggressive tumors.
The anti-invasive effect seen with nontoxic concentrations of ALP may
prove valuable in the treatment of malignant gliomas.
INTRODUCTION
Several characteristic changes on the cell surface occur with
transformation. For tumors of glial linage, these include alter
ations in the expression of growth factor receptors (1-3), as
well as changes in the carbohydrate composition on the plasma
membrane (4). Gliomas are known for their invasiveness into
the normal brain where tumor cells are often found at consid
erable distance from the macroscopic border of the primary
mass (5). A complete tumor resection is therefore difficult and
the prognosis in these patients is generally poor. Drugs that
influence the cell membrane or functional components of the
cell membrane are therefore of considerable interest in cancer
therapy (6).
The antineoplastic drug Et-18-O-CH., belongs to a group of
ALP,4 which are synthetic analogues of the naturally occurring
2-lysophosphatidylcholine
(7). This compound and structural
analogues are known to accumulate at the cell surface, where
they exert their antineoplastic action (8). These drugs are reReceived9/25/90;accepted1/7/91.
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.
' O. E. and R. B. were supported by The Norwegian Cancer Society. Grants
88218 and 89002. M. E. B. was supported by a grant from the Preuss Foundation.
2To whom requests for reprints should be addressed, at The Cade Institute.
Department of Pathology, University of Bergen. Haukeland Hospital. N-5021
Bergen. Norway.
3Current address: Laboratory of Neuro-Oncology Research, Barrow Neuro
logical Institute, 350 West Thomas Road. Phoenix, ÀZ85013-4496.
4 The abbreviations used are: ALP. alkyl-lysophospholipid. DMEM. Dulbecco's modification of minimal Eagle's medium.
garded as biological response .modifiers acting both as immunopotentiators and as cytotoxic agents (9). The ALPs have been
shown to inhibit growth of several different tumors both in vitro
and in vivo (9-11), and exert an anti-invasive effect (12, 13).
In this study, the cytotoxic and anti-invasive effects of Et-18O-CHj were studied on a well-characterized human glioma cell
line (GaMg) (14) as well as on tumor spheroids generated from
3 different primary brain tumor biopsies (15). For this purpose,
an in vitro invasion assay has been developed in which normal
fetal rat brain cell aggregates were used as target tissue for
human gliomas (16-18).
MATERIALS
AND METHODS
Drug. The ether lipid l-octadecyl-2-methyl-rac-3-glycerophosphocholine was obtained from Medmark Pharma Gmbh, Federal Republic
of Germany. The drug was dissolved in 100% ethanol and stored at
4°Cuntil use, at which time serial dilutions in DMEM to the final
concentration were made.
Cell and Tissue Culture. The human glioma cell line GaMg (14)
(passages 25-40) has been characterized before. Briefly, this cell line
was obtained from a 42-year-old female and histologically identified as
a glioblastoma. The biopsy specimen and primary cultures contained
many diploid cells, as analyzed by flow-cytometric DNA measurement.
This changed within one year of subculture, at which time a neartriploid DNA content was found. Karyotyping revealed a mean chro
mosome number of 67.1 ±14.3 (SD). The cell lines were grown in
DMEM supplemented with 10% heat-inactivated newborn bovine
serum, 4 times the prescribed concentration of nonessential amino
acids, 2% L-glutamine, penicillin (100 lU/ml), and streptomycin (100
Mg/ml). The cells were kept in a standard tissue culture incubator at
37°C,with 100% relative humidity, 95% air, and 5% CO2. Cells were
free of Mycoplasma contamination.
Tumor spheroids were initiated using the agar overlay culture method
described by Yuhas et ai ( 19). Briefly, spheroids were formed by seeding
5 x IO6cells in 20 ml of growth medium into 80-cttr agar-coated tissue
culture flasks (Nunc, Denmark). The spheroids were used between days
7 and 14.
Spheroids from Primary Brain Tumor Specimens. Tumor tissue from
patients undergoing craniotomy was cut into small fragments and
cultured according to the method described by Bjerkvig et al. (15).
Briefly, tumor tissue from 3 intracranial tumors, one glioblastoma
multiforme, one anaplastic astrocytoma, and one astrocytoma, was
dissected out at surgery and transferred to DMEM. Macroscopically
representative tissue from the tumor was then selected for tissue culture
and prepared for histopathological diagnosis. The specimens were cut
into 0.5-mm-diameter fragments and placed into culture in 80-cm2
tissue culture flasks (Nunc) base-coated with 0.75% agar (Difco Agar
Noble; Difco Laboratories, Detroit, MI) in DMEM. They were then
cultured until they rounded up and formed spheroids. The volume of
the overlay suspension was 10 ml of DMEM. The tissue was kept in a
standard tissue culture incubator (100% relative humidity, 95% air and
5% CO2). Primary brain tumor spheroids with a diameter of 295 ±5
(SE) Mmwere selected for further use (for further details, see Ref. 15).
Assays for Growth. Cell sensitivity to ALP was tested in microtiter
culture wells using the MTT assay (20, 21). Briefly, cells in exponential
growth phase were collected by trypsinization, and then plated in
microtiter wells (1000 cells/well in 200 M')- The following day, ALP
was added to the wells to achieve the final concentrations (see "Re-
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ALPS INHIBIT GLIOBLASTOMA
suits"). After 5 additional days in culture, 50 p\ of MTT (Sigma
Chemical Co., St. Louis, MO) (5 mg/ml in Hanks' balanced salt
solution) were added to each well, and the plates were incubated at
37°Cfor 2 h. The liquid contents of the wells were emptied by rapid
inversion of the plate, and the wells were dried in a 37°Cdry incubator.
Light mineral oil (Sigma) was added to the wells to solubilize the
formazan salt, and the absorbance above background (wells containing
medium but no cells) was measured at 540 nm in a Titertek MCC340
(Flow Laboratories, McLean, VA). The absorbance from the wells
containing untreated cells was used to calculate the fractional absorb
ance of the wells with treated cells. Experiments were done in replicates
of 6, and the mean absorbance was calculated. The experiment was
repeated twice.
Monolayer growth curves were obtained by seeding 1.75 x IO5cells
in 5 ml into 25-cnr tissue culture flasks (Nunc). After 24 h of culture,
the ether lipid was added to achieve final concentrations of 0.1, 1.0, 10,
and 30 pM. Three flasks in each treatment group were trypsinatcd and
counted electronically, using a Coulter Counter (Coulter Electronics,
Luton, Bedfordshire, United Kingdom).
Spheroid growth curves were obtained by transferring individual
spheroids [diameter. 278 ±3 ¿im(SE)] into 16-mm multiwell dishes
(Nunc) base-coated with 0.5 ml of 0.75% DMEM-agar. The overlay
suspension contained 1.0 ml of growth medium. The ET-18-O-CH,
was added to the wells at 0.1, 1.0, and 10 J/M concentrations. The
spheroid size was recorded regularly during 8 days by measuring 2
orthogonal diameters using an inverted phase-contrast microscope with
a calibrated reticle in the ocular. Spheroid volume was calculated using
the formula for volume of a sphere, and expressed as a fraction relative
to the volume at day 0. Ten spheroids were measured in each group
and the experiments were done in triplicate.
Directional Migration. The area covered by GaMg cells migrating
out from the spheroids explanted on a plastic surface was used as an
index of cell migration (22). Ten spheroids (diameter, 278 ±3 urn (SE)]
in each treatment group were transferred individually into 16-mm
multiwell dishes (Nunc). After 2, 5, 8, and 11 days of culture, the
diameter of each colony was measured in the presence of different
concentrations (0.1, 1.0, 10, and 30 /t\\) of ether lipid. The experiments
were done in triplicate.
Flow Cytometrj. After days 2, 4, 6, and 8 of exponential monolayer
growth, suspensions of the treated and control cells were fixed in icecold 100% ethanol and stored at 40°Cuntil use. The fixed cells were
incubated for 15 min with 0.5% pepsin at 37°C,and the free nuclei
CELL INVASION l.\ tITRO
and allowed lo "mature" for 18 days. At this time, the fetal cells had
formed stable aggregates and developed into morphologically mature
brain cells.
Mature aggregates, with a diameter of 281 ±4 /tm (SE), were
transferred individually into agar-coated 96-well tissue culture dishes
(Nunc). Tumor spheroids with the same size as the aggregates were
also transferred to the wells, and DMEM (containing the actual con
centrations of ALP) was added, giving a total volume of 300 ¿Jin each
well. The normal brain aggregates and the tumor spheroids were then
brought into proximity by use of a sterile syringe. The orthogonal
diameters of the brain aggregates and tumors were then measured. The
co-cultures were fixed after 72 or 168 h for light-microscopic exami
nation and morphometric analyses. In the co-cultures involving the
GaMg glioma cell line, there were 7 observations in each treatment
group at the different fixation points, and the experiments were done
in triplicate. In the invasion experiments using tissue from intracranial
tumors, there were 5 co-cultures in each treatment group.
Light Microscopy. The co-cultures were fixed for 24 h in 2% glutaraldehyde in sodium-cacodylate buffer (300 ±25 mOsm). The speci
mens were then washed in the same buffer, without glutaraldehyde, and
postfixed for l h in 1% OsO4 before serial dehydration in increasing
gradients of ethanol to 100%. Embedding in Epon 812 was performed
using graded mixtures of epon-propylenoxide. The specimens were
polymerized for 48 h at 60°C.
Morphometry. In order to determine the amount of remaining brain
tissue after 72 and 168 h of tumor cell invasion. Chavalieri's principle
for direct estimation of volume from systematically sampled sections
was used (27, 28). Serial semithin sections (1.5 pm) were prepared from
the specimens using a Reichert-Jung Microtome 2040 (Reichert-Jung,
Austria). Every 15th section was sampled starting with one of the 10
first sections that was randomized. The sections were then stained with
toluidine blue for light-microscopic examination. The brain tissue and
the tumor tissue were easily discriminated by the difference in mor
phology. The areas of the tumor tissue and remaining brain tissue were
then measured on each section by morphometry, using a Kontron Image
Analysis system (Kontron, Federal Republic of Germany). The area of
tumor spheroid and brain aggregate measured on each slide, multiplied
by the distance between every sampled section (15 x 1.5 ^m), resulted
in an estimate of the volume for each slice. The total volume of the
tumor tissue as well as the remaining brain tissue was calculated by
summation of all the individual volumes. The coefficient of error in
this method for estimating volumes of sections is accepted to be less
than 5% (27).
Statistics. The Student's t test was used to determine the level of
significance of the difference between individual groups. The doseresponse relationship in the growth curve experiments, and in the
invasion assay, was assessed statistically by regression analysis.
were subsequently washed in 0.9% saline and treated for 5 min with 1
mg/ml ribonuclease dissolved in 0.9% saline (Sigma). Staining of DNA
was performed with a mixture of ethidium bromide and mithramycin
(for details, see Ref. 23). The relative cellular DNA content was
measured with a Phywe ICP-22 pulse cytophotometer (Ortho Instru
ments, Westwood, MA). Normal human lymphocytes were used for
instrument adjustment and calibration. Cell cycle analysis was per
RESULTS
formed using a planimetrie method originally described by Baish et al.
(24); 15,000 cells were analyzed in each of 3 replicate observations in
MTT—Assay and Tumor Growth. The GaMg cells showed a
the different treatment groups (coefficient of variation <5%).
dose-dependent inhibition of growth by ALP after 6 days in the
Assay for Tumor Cell Invasiveness. A standardized in vitro invasion
MTT assay; the 90% inhibitory concentration was 50 MM(Fig.
assay was used to study the anti-invasive effects of Et-18-O-CH., (17,
1). Cells in the growth curve assay were in exponential phase
18). Fetal rat brain cell aggregates were used as a normal target tissue
for 4 days, showing 13% (P < 0.025) and 35% (P < 0.005)
for the tumor spheroids. Such brain aggregates have a well-developed
neurophil with mature neurons, astrocytes, and oligodendrocytes. My- growth inhibition by 10 and 30 MMALP, respectively (Fig. 2).
No effect from 0.1 or 1.0 MMtreatment was detected.
elinated axons and synaptic complexes are frequently observed in the
Dose-dependent inhibition of spheroid growth was detected
aggregates thus showing many similarities to the normal adult brain
(16, 25). The procedure for obtaining fetal rat brain aggregates has
using 10 and 30 MMALP (Fig. 3). These concentrations of ALP
been described in detail elsewhere (16). Briefly, 18-day-old fetuses from
also elicited severe damage to the integrity of the spheroids
pregnant BD IX rats (26) were removed by cesarean section. The brains
when examined by phase-contrast microscopy. The spheroids
of the fetuses were carefully dissected out, minced, washed 3 times in
had a ruffled surface appearance and pronounced cell shedding
phosphate-buffered saline, and serially trypsinized. The resulting singleinto the medium. Spheroids treated with ALP at 0.1 and 1.0
cell suspension was then centrifuged at 1500 rpm for 5 min and
MM,or the control wells maintained smooth external surfaces
resuspended in DMEM; IO7cells were then seeded into 24-well dishes
and did not show any signs of cytotoxicity.
(Nunc) previously coated with 0.5 ml of DMEM-agar. The cells were
Cell Cycle Effects. Treatment with 30 //M Et-18-O-CH,
allowed to reaggregate for 2 days. Thereafter, the aggregates were
transferred to a larger number of wells (5 to 7 aggregates in each well) caused an accumulation of cells in the G2M compartment of
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ALPS INHIBIT GLIOBLASTOMA
1.00
0.10
0.03
Dose (uM)
Fig. 1. Dose-response relationship
analyzed in the MTT assav.
of treated GaMg
monolayer
culture as
3.0Conlrol
2.5 -
0.1
l ^M
I
2.0-
lOuM
30 uM
CELL INVASION 1\ VITRO
aggregates consisted of a loosened neuropil. In co-cultures
treated with 0.1 and 1.0 ^M ALP, the brain aggregate was
intact, lacking only the peripheral fibrous layer. The center of
the aggregate was preserved, with no change in morphology
from control aggregates (Fig. IB); 10 n\i ALP had serious toxic
effects on both the normal and the tumor tissue (Fig. 1C).
Necrosis developed after 72 h and increased in magnitude
within 168 h.
All the primary brain tumor biopsies progressively invaded
and destroyed the brain tissue. In the glioblastoma multiforme,
extensive destruction of the target tissue was evident within 72
h, with an almost total destruction of brain tissue after 168 h
in the control co-cultures (Fig. 8). The lower grade brain tumor
spheroids invaded and destroyed the brain tissue to a lesser
extent, as reported previously (17) (Fig. 8).
The invasion of the glioblastoma multiforme was severelyinhibited by 1.0 ¡IM
ALP. The treatment preserved the remain
ing brain volume by 31% after 72 h (P < 0.05). After 168 h,
such treatment caused a 7-fold preservation of normal brain
tissue (P < 0.025) as compared with untreated co-cultures (see
Figs. 8 and 9).
The reduction in invasiveness was less pronounced for the
less invasive, lower grade tumors. In treated co-cultures of the
4.0-1
o
3.5-
>
§
3.0-
Time (Days)
Fig. 2. Growth curves of GaMg cells during continuous exposure to ALP.
Bars. SEM from triplicate measurements. No reduction in exponential cell growth
was observed at ALP doses lower than 10 »JM.
•a
JS
o
OÃ-
the cell cycle (21.9% as compared with 15.3% for control
cultures: P < 0.005) and a concomitant significant reduction of
cells in the G, phase of the cell cycle (from 68 to 59%, P <
0.005) (Fig. 4). This effect was evident by the second day of
drug exposure and persisted through the 8 days of
measurement.
Directional Migration. Treatment with ALP at low concen
trations (0.1 and 1.0 /UM)did not cause any effect on cell
outgrowth from the spheroids (Fig. 5); 10 uM ALP caused a
27% reduction in outgrowth area after 11 days of drug exposure
(P < 0.005), whereas 30 ^M drug reduced the colony area to
40% of controls (P < 0.0005).
Anti-Invasive Kffcct. In co-cultures between GaMg spheroids
and brain aggregates, progressive destruction and invasion of
the brain aggregate by the glioma cells occurred. After 168 h of
1.0 MMALP treatment, the volume of the intact brain aggregate
was 90% larger than that in control co-cultures. Dose-depend
ent preservation of brain aggregate was evident after 72 h,
which reached statistical significance (P < 0.005) at 168 h (Fig.
6A). There was no significant difference in glioma spheroid
volumes after 72 or 168 h with the concentrations used in these
experiments (Fig. 6Ä).
In control co-cultures, brain aggregates were severely de
stroyed by invading glioma cells within 168 h (Fig. 7.4). The
structure of the brain aggregate was disrupted, the outer fibrous
layer of glial-like cells was lost, and the remaining core of the
12345678
Time (days)
Fig. 3. Growth of GaMg multicellular tumor spheroids treated with ALP.
Spheroid growth is recorded relative to the calculated volume at day 0, using the
formula for the volume of a sphere. Bars, SEM from 10 replicate spheroids at
each treatment dose.
30 uM
1.0 uM
G,s c;2M
UNA content
Control
Fig. 4. Cell cycle distribution of GaMg monolayer cultures as analyzed by flow
cytometry after 2 days of treatment with ALP. showing an accumulation of cells
in the G;M phase, and concomitant reduction of cells in (he G, phase. Analysis
at days 4. 6. and 8 rc\ caled identical effects of ALP.
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Al.l'S IMIIBII
(,l IODI ASIOMA
anaplastic astrocytoma, the brain tissue was 53% (P < 0.05)
larger after 168 h (Fig. 9), whereas treatment of the astrocytoma
preserved the amount of target tissue by 34% (not significant;
see Figs. 8 and 9).
CELI. INVASION /.Y IÕTRO
V
DISCUSSION
Malignant gliomas are characterized by a highly invasive
behavior in the brain (5, 29). The lack of effective treatment of
such tumors implies a poor prognosis for the patients, and new
therapeutic strategies are therefore needed. ALP is known to
cause cytostatic and cytotoxic effects in both rat and human
gliomas (10, 11). The present study confirms these results
showing reduced cell growth after ALP treatment. In addition,
it is shown that ALP is an anti-invasive drug not only for
glioma cell lines but also for biopsies obtained from primary
human brain tumors.
Treatment with ALP led to a dose-dependent growth inhibi
tion of GaMg human glioma cells in monolayer culture. The
MTT assay showed the 90% inhibitory concentration to be
approximately 50 /¿M,
and in the growth curve studies, 30 /IM
ALP resulted in 35% growth reduction after 4 days of drug
exposure during exponential growth. Prior studies against var
ious tumor cell lines have shown that concentrations above 20
/¿M
are required for growth inhibition in monolayer culture (11,
30). In addition, short-term treatment (<2 h) with ALP does
not exert an antiproliferative effect (10).
Tumor spheroid growth curves showed that 10 /¿MALP
inhibited growth after 6 days in tissue culture and had a dele
terious effect on the spheroid morphology. The increased drug
* ItfSWR
' *t«V?WV%ÄVl
B
40 -i
2
4
6
Time (days)
8
10
12
Fig. 5. Monolayer outgrowth of GaMg spheroids after addition of ALP. Bars,
SEM from 6 replicate spheroids at each treatment dose.
Fig. 7. Photomicrographs of co-culture experiments between GaMg spheroids
and rat brain aggregates. A, control co-cultures showing extensive invasion and
degradation of the brain tissue (B'). B, co-culture treated with 1.0 ^M ALP. The
brain aggregate (B') is well preserved. C. co-cultures treated with toxic doses of
ALP (10 tiM) B'. remnants of the brain aggregate: bars. 50 ¿im.
(I.(K)K -j
Fig. 6. Effect of ALP treatment on volumes of rat
brain aggregates (A) and GaMg tumor spheroids in
co-culture (/?). Volumes represent values calculated
from reconstructed image analysis of serial sections.
Bars, SEM from 7 replicate co-culture experiments in
each group.
Time (hours)
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ALPS INHIBIT GLIOBLASTOMA
CELL INVASION /.V IITRO
*
B
_,>v»"
^^
' ;
•
•:•*.
•'*
Fig. 8. Photomicrographs of co-culture experiments involving the spheroids from primary brain tumor specimens. A. spheroid from glioblastoma multiforme
confronted with brain aggregate (B') after 72 h of incubation. B, co-culture between glioblastoma multiforme and brain tissue (B'}. treated with 1.0 JJ.MALP for 72
h. C, control co-culture between a spheroid originating from an anaplastic astrocytoma and a fetal rat brain aggregate (B') after 168 h of incubation, D, co-culture
between anaplastic astrocytoma spheroid and brain aggregate (B') after 16X h of incubation, treated with 1.0 /IM ALP (bars. 5
sensitivity of GaMg in spheroid culture compared with monolayer culture may be related to the increased cell-to-cell inter
actions brought into play by 3-dimensional culture. Since the
mechanism of action of ALP is dependent on integration of the
drug in the plasma membrane of the target cells (9), it is
reasonable to speculate that monolayer cultures provide a less
susceptible target for the drug. This is not a novel finding, as
differential sensitivity of tumor cells based on assay methods
has been reported previously (31-33). In an in vivo study,
treatment by ALP caused an accumulation of the drug in the
tumor tissue, as compared with normal tissue (34). If this is the
case, in 3-dimensional tissue culture is presently not known.
A concentration of 30 n\\ ALP caused an accumulation of
cells in the G2M phase of the cell cycle after 2 days' exposure
0.11(17 :
Fig. 9. Measured volumes from serially sectioned
co-cultures of a glioblastoma multiforme, an anaplas
tic astrocytoma. and an astrocytoma. Bars, SEM from
5 replicate co-cultures in each group.
Ghoblastoma multiforme
72 hours
I68hours
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ALPS INHIBIT (¡I.IOBIASTOMA CELL INVASION l.\ \ÕTRO
in monolayer culture. Cytotoxic effects have been associated
with increased lipid fluidity (35), the formation of blebs and
holes in the plasma membrane (8), and modulation of the
protein kinase C system (36, 37). ALP does not seem to inhibit
microtubule assembly or reassembly in MO4 mouse fibrosar
coma cells (12). An induction of multinucleated cells by ALP
treatment in sensitive cell lines has been found previously (38).
The accumulation of cells in G:M phase of the cell cycle may
be due to an interference of ALP with the plasma membrane
with a retarded cell division as the result.
The most profound and novel observation from our studies
is the effect of nontoxic concentrations of ALP on the invasive
behavior of glioma cells into an in vitro model of brain tissue.
Since complete surgical resection of astrocytomas and glioblastomas is not feasible, and since these tumors are highly refrac
tory to chemotherapy and radiation, agents that block other
phenotypic traits of these malignant cells may have exciting
potential as new treatment modalities against these neoplasms.
The invasion of tumor spheroids into fetal rat brain aggre
gates was inhibited after 72 h of co-culture, but the magnitude
of inhibition did not reach statistical significance until 168 h.
This may be due to a slow integration of drug molecules into
the plasma membrane (9), which would lead to a delayed action
of ALP. The retarded anti-invasive effect may also arise from
a technical limitation of the assay, in which more extensive
invasion by GaMg control co-cultures is necessary for differ
ences between control and treated dishes to become significant.
Other authors have described a total prevention of tumor cell
invasion into chick heart fragments after incubation with ALP
(12, 13). The discrepancy between the demonstration of early
and complete inhibition of invasion in their studies and the
delayed efficacy in our report may be attributable to differences
in the 2 model systems used. The mechanisms of glioma cell
invasion into fetal rat brain aggregates, which represents a tissue
of neuroectodermal origin, may be expected to be different from
those required for the invasion into precultured chick heart
fragments. Furthermore, by virtue of the natural invasiveness
of glioma cells into brain and the extraordinarily low incidence
of glioma metastasis extracranially (29), brain aggregate may
be a more relevant model for the investigation of processes
operative in the clinical setting of gliomas. Invasion may be due
to specific interactions between cells and the interstitial matrix
of the invaded tissue; the composition of the matrix would be
markedly different in chick heart versus rat brain. Finally,
different tumors may show various responses to anti-invasive
therapy (13).
Additional impetus to the potential utility of ALP as an antiinvasive therapy against human gliomas is gained from our
results using primary glioma spheroid cultures in the invasion
assay. The profound inhibition of the glioblastoma cell invasion
at a concentration of 1.0 MMsuggests that ALPs induce pertur
bations of the cell membrane, thus interfering with the invasive
phenotype of glial cell tumors. Because of the relationship
between the invasiveness of the glial tumor, and the ability of
ALP to inhibit the invasion for the tumors studied, we suggest
that this property is directly interfered with by ALP. The
biochemical origin of this anti-invasive effect of ALP should be
further exploited for the development of therapy and to better
understand glioma progression.
ALP at a concentration of 1.0 MMdid not inhibit cell migra
tion (spreading of spheroids onto monolayer), but did inhibit
invasion after 168 h of co-culture. It is likely, therefore, that
ALP exerts direct anti-invasive effects on glioma cells inde
pendent from influences on motility (migration) and prolifera
tion. The mechanism(s) responsible for the anti-invasive effect
are not known, but alterations in the cell surface carbohydrates
(4, 39, 40) could play a major role, as carbohydrates are found
in relatively high concentrations on the cell surface in the form
of glycopeptides, glycosylated proteins (cell recognition mole
cules), and glycolipids (gangliosides). All these molecules are
thought to be important for cellular interactions and recogni
tion (41, 42).
It is known that malignant cells have a carbohydrate com
position different from that of nonmalignant cells, and that
ALP treatment changes the carbohydrate moieties on the cell
membrane (4, 43). If a change in carbohydrate composition is
associated with invasiveness, this may explain the transition of
noninvasive, transformed cells to invasive cells by ALP treat
ment, as have been documented in a previous study (4). Changes
in carbohydrate composition in embryonal cells treated by ALP
may also explain the limited therapeutic index seen in our
study, in which 10 MMof this drug destroyed the fetal rat brain
aggregates. In contrast to this, ALP has been shown not to
interact with cellular DNA and has no mutagenic effects (44).
In addition, limited toxic effects have been observed from ALP
during Phase I clinical trials (9, 45).
ALP has been shown to penetrate the blood-brain barrier
more readily than other cytostatics (46). Furthermore, there is
evidence for an enrichment of this compound in tumors in vivo,
as compared with normal tissue (34). This makes the drug
interesting as a potential agent in brain tumor therapy. The
relatively large inhibition of invasion achieved by ALP at nontoxic doses suggests a potential use for ALP, or its derivatives
in the treatment of malignant gliomas.
ACKNOWLEDGMENTS
The authors are greatly indebted to Dr. O. D. Laerum for valuable
advice and criticism.
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Effect of Alkyl-lysophospholipid on Glioblastoma Cell Invasion
into Fetal Rat Brain Tissue in Vitro
Olav Engebraaten, Rolf Bjerkvig and Michael E. Berens
Cancer Res 1991;51:1713-1719.
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