Download Cytotoxicity of Alkyl-lysophospholipid Derivatives

[CANCER RESEARCH 43, 541-545, February 1983]
0008-5472/83/0043-0000502.00
Cytotoxicity of Alkyl-lysophospholipid Derivatives and Low-AlkyI-Cleavage
Enzyme Activities in Rat Brain Tumor Cells
Wolfgang E. Berdel, 1 Eva Greiner, 2 Ulrich Fink, Dimitrios Stavrou, Anneliese Reichert, Johann Rastetter,
Dennis R. Hoffman, 3 and Fred Snyder 4
Division of Hematology and Oncology, Department of Medicine I, Technical University [W. E. B., E. G., U. F., A. R., J. R.], and Department of Functional Pathology,
Ludwig-Maximilians-University [D. S.], 8000 Munich, Federal Republic of Germany, and the Medical and Health Sciences Division, Oak Ridge Associated
Universities, Oak Ridge, Tennessee 37830 [D. R. H., F. S.]
ABSTRACT
Alkyl-lysophospholipids (ALP) and related derivatives inhibited the in v i t r o incorporation of [3H]thymidine into seven
different permanent cell lines derived from rat brain tumors.
The cytostatic effect of ALP was dependent on dosage and
incubation time. Naturally occurring 2-1ysophosphatidylcholine
did not exhibit cytostatic effects; under these conditions, the
incorporation rates of [3H]thymidine were generally more than
100% of the controls. The trypan blue dye exclusion test,
which was used to assess severe cell damage, correlated with
the extent that [3H]thymidine incorporation was inhibited by
ALP.
Preincubation of ALP ( r a c - 1 -octadecyl-lyso-glycero-3-phosphocholine) for more than 8 min with a tetrahydropteridinedependent O-alkyl cleavage enzyme preparation from rat liver
microsomes destroyed almost all of the cytotoxic properties of
ALP when tested at a concentration that previously inhibited
tumor growth by more than 50%. [3H]Thymidine incorporation
rates were greater than 1 00% for astrocytoma cells incubated
with ALP after exposure to the alkyl cleavage enzyme.
Comparison of the microsomal activities of the tetrahydropteridine-dependent alkyl-cleavage enzyme present in astrocytoma 78-FR-G-299 cells and the pleomorphic glioma 78-FRG-21 9 / $ 4 cells to that found in normal skin fibroblasts and rat
livers revealed a markedly reduced activity in the neoplastic
cell lines. Moreover, those tumor cells that were more resistant
to ALP cytotoxicity (pleomorphic glioma, 7 8 - F R - G - 2 1 9 / S 4 )
had a 3-fold higher tetrahydropteridine-dependen t cleavage
activity than a more cytotoxic sensitive line (astrocytoma cells,
78-FR-G-299). Our results indicate that the Iow-alkyl-cleavage
enzyme activities in these neoplastic cells in comparison to
normal cells might be a factor in explaining the relatively high
cytotoxicity of ALP in tumor cells.
INTRODUCTION
ALP 5 and related derivatives (synthetic analogues of naturally
Received November 19, 1981; accepted November 4, 1982.
1 Supported by Deutsche Forschungsgemeinschaft Grants Be 822/2-2 and
Sta 134/2. To whom requests for reprints should be addressed: Division of
Hematology and Oncology, Department of Medicine I, Technical University,
Ismaninger Str. 22, 8000 Munich 80, Federal Republic of Germany.
2 In partial fulfillment of the M.D. degreeof the Technical University of Munich.
3 Fellowship supported by Grant CA 09336 awarded by the National Cancer
Institute to the University of Tennesseeand the Oak Ridge National Laboratory.
4 Supported by the Office of Energy Research, United States Department of
Energy (Contract DE-AC05-760R00033), and the American Cancer Society
(Grant BC-70M).
5 The abbreviations used are: ALP, alkyl-lysophospholipids; Pte.H4, tetrahydropteridine; ET-18-H, rac-l-octadecyl-2-hydride-glycerophosphocholine; ET18-OH, rac-l-octadecyl-lyso-glycerophosphocholine; ET-18-OCH3,rac-l-octadecyl-2-methoxy-glycerophosphocholine; ET-12-H, rac-l-dodecyl-2-hydride-
occurring 2-LPC) represent a new kind of antitul"nor agent with
immune-modulating potential (1 5). It has been shown that ALP
inhibits the growth of human leukemic cells (1, 24), hypernephroma cells (6), cells from various urological tumors (7), and
different human malignoma cells (4) in vitro. In contrast, human
embryonic fibroblasts (7) as well as bone marrow cells (1, 1 4)
are less affected by the same doses of ALP that are toxic to
tumor cells. In vivo, ALP is also able to prevent or retard tumor
growth and to inhibit metastasis in several syngeneic tumor
models in mice (3, 5, 1 5, 23).
The mode of action for the antineoplastic activity of ALP has
been explained as a direct cytotoxic activity on tumor cells and
on their ability to generate tumoricidal macrophages (5, 8, 9).
In contrast to normal cells, neoplastic tissues are characterized
by the lack of a Pte.H4-dependent ether cleavage enzyme
activity (1 8). Therefore, ether-linked lipids are not catabolized
in tumors as they are in normal cells, and the accumulation of
the " l y s o " type of ether phospholipids can interfere with vital
pathways of phospholipid metabolism of the cell (1 4). It appears
likely that the direct cytotoxic effect of ALP is dependent on
the ether-linked alkyl moiety of the glycerophosphocholine lipid
and the lower activity of the O-alkyl cleavage enzyme system
in tumor cells (1 8).
Our study describes the cytostatic and cytotoxic effect of
ALP in various rat brain tumor cells and deals with the question
of whether 2-LPC, the ester phospholipid analogue, is less
cytotoxic than the ether-linked analogue. Furthermore, the
influence of pretreatment of the O-alkyl lipid analogues with
the Pte. H4-dependent O-alkyl cleavage enzyme on the tumor
cytotoxicity of ALP has been tested. Data were also obtained
that demonstrate that the cytotoxicity of ALP is inversely related
to the microsomal activities of the cleavage enzyme.
MATERIALS AND METHODS
ALP. ALP (ET-18-H, ET-18-OH, ET-18-OCH3, and ET-12-H) were
kindly provided by Dr. H-U. Weltzien (Max-Planck-lnstitute for Immunobiology, Freiburg, Federal Republic of Germany). Details about their
chemical structure and synthesis are given elsewhere (2, 4, 15, 25).
The ALP and the naturally occurring ester analogue, 2-LPC (ES-16/
18-OH; Fa. Roth, Karlsruhe, Federal Republic of Germany) were dissolved in RPMI Medium 1640 (GIBCO No. 240; Gibco, Glasgow,
Scotland), containing 10% fetal calf serum (GIBCO No. 176), sterilized
by micropore filtration (0.22/~m; Millex; Millipore, Molsheim, France),
and stored at - 2 0 ~.
Cells. Seven permanent cell lines of malignant rat brain tumors
were used in this study. The tumors were experimentally induced in 6-
glycerophosphocholine; 2-LPC, (ES-16/18-OH), rac-l-hexadecyl/octadecyllyso-2-glycerophosphocholine, i.e., 2-1ysophosphatidyl-choline; RPMI, Roswell
Park Memorial Institute.
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W. E. B e r d e l e t al.
month-old Fischer (F344) rats by N - m e t h y I - N - n i t r o s o u r e a in the drinking water. Details of induction and cultivation of these cells have been
published (20, 22); the histological origin of each cell line is listed in
Table 1, whereas the code designating each line is explained elsewhere
(21). All cell lines have gone through more than 50 passages. In order
to prepare single-cell s u s p e n s i o n s from the monolayers, supernatants
were removed from the flasks (75 sq cm; Falcon Plastics, Oxnard,
Calif.), and the monolayers were overlaid with 3 to 4 ml of a 0.9%-NaCI
solution:trypsin:EDTA solution. Cells were incubated at 37 ~ for 5 rain
and then washed from the surface with a jet of medium (RPMI Medium
1 6 4 0 supplemented with 10% fetal calf serum, containing 50 units
penicillin and 50 #g streptomycin per ml). The cells were gently pipeted
into tubes, centrifuged at 2 5 0 x g for 10 rain, and resuspended in
medium to a final concentration of 1 x 105 c e l l s / m l . Viability of the
cells, based on trypan blue dye exclusion (11 ), was greater than 95%.
[ 3 H ] T h y m i d i n e I n c o r p o r a t i o n . The cytostatic effect of the substances tested was based on a decrease of [3H]thymidine incorporation
into the tumor cells. A 100-/~1 aliquot of the tumor cell suspension (1
x 105 c e l l s / m l ) was placed into each well of a Microtiter plate (Greiner,
Nurtingen, Federal Republic of Germany), and the ALP (100/~1) was
then added at various concentrations in triplicate. Controls contained
100 #1 of pure medium instead of the test substance. Plates were
incubated at pH 7.2, at 37 ~ in an atmosphere of 5% CO2 and high
humidity for 24, 48, 72, or 96 hr. All cultures were pulsed for 24 hr
with 0.5/~Ci of [6-3H]thymidine per well (5 C i / m m o l ; Amersham and
Buchler, Braunschweig, Federal Republic of Germany). The samples
were harvested and radioassayed after freezing and thawing as described previously (7); standard deviations were less than -4-_10%.
E n z y m a t i c Studies. Microsomes were isolated from the livers of
Fischer (F344) rats (17). The ALP used as substrate was ET-18-OH.
The standard assay system (16) was modified as follows. Fifty/~g of
the microsomal protein suspended in 0.25 M sucrose were added to
the system containing 30, 40, 50, or 60 #g of ET-18-OH, Pte.H4 (1
mM), reduced glutathione (5 raM), and (NH4)2SO4 (5 raM) dissolved in
borate (0.1 mM):NaOH buffer at pH 8.8; the final volume was 1 ml.
Samples were incubated for 4, 8, 15, 30, 60, or 90 min at 37 ~ in a
shaking water bath. The reaction was stopped by placing the samples
in an ice bath. O-Alkyl clevage was tested by comparing the cytostatic
effect of enzymatically treated ET-18-OH with that of unreacted ET-18OH. An aliquot of 100 #1 of the tumor cell suspension (1 x 10 s c e l l s /
ml) was incubated for 24 hr under the conditions mentioned earlier with
100 #1 of the incubation solution in triplicate for each concentration
Table 1
Influence of different ALP compounds on [3H]thymidine incorporation into 7 cell lines of rat brain tumors
[3H]Thymidine incorporation
(% of control)
Histology
78-FR-G-299a (astrocytoma)
78-FR-G-284 (mixed glioma)
78-FR-G-344 (mixed glioma)
79-FR-G-41 (astrocytoma)
78-FR-G-219/$1 (pleomorphic
glioma)
78-FR-G-219/$2 (pleomorphic
glioma)
78-FR-G-219/$4 (pleomorphic
ALP
Dosage
(/~g/ml)
Incubation for 1
day
Incubation
for 2 days
ET-18-H
10
20
40 b
20
28
14
ET-18-OH
10
20
38
24
32
17
ET-18-H
10
20
81
36
78
26
ET-18-OCH3
10
20
71
32
30
15
ET-18-H
10
20
130
64
80
72
ET-18-OH
10
20
98
109
90
70
10
20
61
43
69
30
ET-18-OCH3
10
20
55
42
36
17
ET-18-H
10
20
96
65
52
16
ET-18-OCH3
10
20
78
48
18
23
ET-18-H
10
20
75
71
52
53
ET-18-OCH3
10
20
>100
>100
62
60
ET-18-H
10
20
30
102
72
34
41
10
20
30
84
65
22
47
39
22
ET-18-H
glioma)
ET-18-OCH3
46
19
a For explanation of the code designating the cell lines see Ref 21.
b Mean values of triplicate cultures. For culture conditions and standard deviations, see "Materials and
Methods."
542
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Cytotoxic Effect of Ether Lipids in Rat Brain Tumor Cells
and incubation time. Control suspensions were incubated with the
same concentrations of unreacted ET-18-OH (30, 40, 50, or 60/~g/
ml) dissolved in sucrose-supplemented borate buffer containing Pte.
H4, reduced glutathione, and (NH4)2SO4. In order to assess the effect
of rat microsomes on tumor cell growth, the control cells were incubated in medium (RPMI Medium 1640 with 10% fetal calf serum)
containing supplemented borate buffer and an aliquot of the alkyl
cleavage assay system minus ET-18-OH. Cytostasis was measured by
[3H]thymidine incorporation; standard deviations were less than _+10%.
In another set of experiments, the activity of the Pte.H4-dependent
alkyl clevage enzyme was assayed in microsomes as described before
(16), except that 1-[9,10-3H]octadecylglycerol (20 #Ci//zmol) served
as substrate. Microsomes were prepared from near-confluent cultures
of normal human skin fibroblasts (Detroit 551), rat astrocytoma cells
(78-FR-G-299), rat pleomorphic glioma cells (78-FR-G-219/S4), and
the liver of a male 180-g CDF rat. Microsomal protein (10 to 20/~g)
was incubated with the reaction mixture (16) for 10 min. The product,
fatty aldehyde, was isolated chromatographically and then analyzed by
liquid scintillation spectroscopy (19). Alkyl cleavage of 1-[9,10-3H] octadecylglycerol was linear with rat liver microsomes up to 20 /~g
protein and over an incubation time of 20 min.
Microsomal protein concentrations were determined according to
the procedure of Lowry et al. (13).
150
/ ~
p-
5O
A
Z
0
I
+
#
:
+
#
i
:
k--
0
Z
RESULTS
IJ.J
Z
Table 1 shows the cytostatic potency of the most effective
ALP analogues (i.e., 18-carbon alkyl chains) with 7 established
rat brain tumor cell lines. No correlation was found between
resistance against ALP and the number of cell passages (data
not shown). The cytostatic effect of ALP increased with dose
and incubation time (Table 1 ) but varied considerably between
different cell lines.
The dose-response relationship for the various ALP analogues is shown in Chart 1. Cells of astrocytoma 78-FR-G-299
were incubated with 3 types of ALP and 2-LPC (ES-1 6 / 1 8 - O H )
for 1 , 2 , 3, and 4 days. Naturally occurring 2-LPC was ineffective and in some instances even stimulated the [3H]thymidine
incorporation rate above control values (Chart 1 A). The 3 ALP
samples revealed a clear dose and time dependence of antitumor action. After 3 days of cultivation of the tumor cells with
ALP concentrations of 20 #g/ml, the [3H]thymidine incorporation was less than 1 5% of the controls. However, there were a
few exceptions from the dose and time dependence of the
ALP-induced cytostasis that lack an explanation (Table 1 ; see
79-FR-G-41 and 7 8 - F R - G - 2 1 9 / S 2 ; also Chart 1).
The trypan blue dye exclusion test was performed to make
sure that treatment with ALP not only decreased the proliferation of tumor cells but actually caused the death of the cells.
Viability expressed as percentage of control correlated well
with the corresponding values obtained for the inhibition of
[3H]thymidine incorporation (Table 2). Viability of cells treated
with ALP also proved to be dependent on incubation time
(Chart 2).
The requirement of the O-alkyl bond for expression of the
antitumor properties of ALP becomes obvious by comparing
[3H]thymidine incorporation before and after the incubation of
ET-18-OH with a microsomal preparation of the O-alkyl cleavage enzyme, which is a Pte.H4-dependent monooxygenase
(Chart 3). Two different sublines of an astrocytoma and 2
different microsomal preparations were tested. The values at
an incubation time of 0 min represent the cytostatic effect of
unreacted ET-18-OH. Four concentrations of ALP were incu-
-II---
50.
I...........
"~l--........ u..-f /
J
~--"~.......
'lk- ......
1
~
D
C
2
3
4
1
2
3
4
DAYS
OF CULTIVATION
Chart 1. [3H]Thymidineincorporation into cells from an astrocytoma line (78FR-G-299) during incubations for 24, 48, 72, or 96 hr with 3 types of ALP or 2LPC (ES-16/18-OH) at different concentrations. See "Materials and Methods"
for culture conditionsand standarddeviations. Incubations:A, ES-16/18-OH; B,
ET-18-OH; C, ET-18-OCH3; D, ET-18-H. Concentrations(#g/ml): 0, 5; m, 10;
A, 20.
bated for different times with a suspension of rat liver microsomes containing the Pte. H4-dependent O-alkyl cleavage enzyme as described in "Materials and Methods." Tumor cells
were then incubated with the enzymatically treated ET-18-OH.
The percentage of metabolized substrate and the kinetics of
the enzymatic reaction are reflected by the alteration of the
antitumor effect of ALP during the incubation period. At a
concentration of 30 # g / m l of ET-18-OH, an incubation of 8 min
was sufficient to remove all of the tumor cytotoxicity associated
with the ALP based on [3H]thymidine incorporation. Optimal
cleavage activity was observed after 30 to 60 min of incubation
with a concentration of 40 #g ET-18-OH per ml and an enzyme:substrate relationship of 50/~g protein:40/~g substrate.
At a substrate concentration of 60 # g / m l , very little effect on
[3H]thymidine incorporation was observed, probably because
of an insufficient amount of enzyme for the large amount of
substrate incubated. Control samples containing boiled microsomes (data not shown) did not alter the [~H]thymidine incorporation by the cells.
Assays of the Pte. H4-dependent alkyl monooxygenase activity in microsomes isolated from cells that are highly sensitive
(astrocytoma cells) to ALP cytotoxicity and cells that are more
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W. E. B e r d e l e t al.
Table 2
Correlation between [~H]thymidine incorporation and viability of rat brain tumor cells as tested with trypan
blue dye exclusion after incubation with ALP or 2-LPC (ES-16 /18-OH)
78-FR-G-219/$1 a
Substance
[3H]Thymidine incorporation c
Viability c
Control
ET-18-H
ET-18-OH
ET-18-OCH3
ET-12-H
ES-16/18-OH
100
73
56
57
71
66
1O0
54
59
34
62
74
78-FR-G-219/$2 a
[3H]Thymidine incorporation
1O0
84
75
72
95
94
78-FR-G-344 b
Viability
[aH]Thymidine incorporation
Viability
100
64
73
84
90
95
100
7
24
47
89
93
100
4
20
20
40
90
a After an incubation period of 48 hr with concentrations of 20 #g/ml.
b
After an incubation period of 24 hr with concentrations of 50/~g/ml.
c [3H]Thymidine incorporation and the number of viable cells are expressed as percentage of the control
samples, i.e. cells incubated without the test phospholipids.
IA
100
/~'--
~100
~
g
~
B
z
o
rr
o
13..
(3:
o
(J
z- 6
LLI -~-,
r'~ u
>-1--'I - - ~,_
50
~'o
(,1
~
'k
t
......
x
I
I
1
2
DAYS OF I N C U B A T I O N
Chart 2. Correlation between [3H]thymidine incorporation and viability (based
on trypan blue dye exclusion) of pleomorphic glioma cells (78-FR-G-219/S1)
after 24- or 48-hr incubations with 20-#g/ml concentrations of ALP: 0, ET-18-H;
II, ET-18-OCH3.
(pleomorphic
glioma
c e l l s ) to A L P
cytotoxicity
vealed that the latter had a 3 times higher O-alkyl
re-
cleavage
a c t i v i t y t h a n d i d t h e m o r e s e n s i t i v e t y p e c e l l s ( T a b l e 3). T h e
much higher levels of Pte. H4-dependent
ties associated
with normal
these experiments.
the human
alkyl cleavage activi-
c e l l s (1 8 ) w a s a l s o o b s e r v e d
Microsomal
skin fibroblasts
in
preparations from rat liver and
had alkyl cleavage
were 32- and 19-fold (respectively)
the astrocytoma cells.
3b 6b 9b
'~
-
o
4
8
15 30 6o 9o
"
TINE OF INCUBATION (mini
Chart 3. Comparison of the effect of unreacted ET-18-OH (0 min) and enzymatically treated ET-18-OH on [3H]thymidine incorporation into 2 different sublines (A and B) of an astrocytoma. ET-18-OH was incubated with the rat liver
microsomal protein and O-alkyl cleavage enzyme reaction mixture for O, 4, 8,
15, 30, 60, or 90 min. See "Materials and Methods" for details. Cells were
incubated with 4 different concentrations of reacted and unreacted ET-18-OH.
Values are means of triplicate cultures; standard deviations are given in
"Materials and Methods." Concentration of ET-18-OH: ~g/ml): V, 3 0 ; . , 40;
0 , 50; A, 60.
I
resistant
~
TINE OF INCUBATION (rain)
:t
<
:-:--rZ
7
i
Table 3
Activity of Pte. H4-dependent alkyl cleavage enzyme in microsomes from normal
human skin fibroblasts (Detroit 551), rat astrocytoma cells (78-FR-G-299), rat
pleomorphic glioma cells (78-FR-G-219 / $4), and CDF rat liver
Specific activity a
(pmol//~g protein/min)
Microsomal source
Rat liver
Skin fibroblasts
Astrocytoma
Pleomorphic glioma
8.85
5.40
0.28
0.88
_+ 0.49 b
___0.91
_+ 0.07
+_ 0.14
a Activity represents cleavage of 1-[9,10-3H]octadecylglycerol as described
in "Materials and Methods."
b Mean ___S.D. of 2 separate microsomal preparations, each done in duplicate.
activities that
h i g h e r t h a n t h a t f o u n d in
s h o w e d v a r y i n g d e g r e e s o f s e n s i t i v i t y to A L P c y t o t o x i c i t y ( T a b l e
1). A c l e a r d o s e - r e s p o n s e
relationship
was found
(Chart
1),
and the cytostatic effect of ALP increased with incubation time
DISCUSSION
( T a b l e 1 ; C h a r t 1). O n l y t h e e t h e r - c o n t a i n i n g
sessed the cytotoxic
Our data indicate that ALP inhibit the incorporation of [3H]thymidine into rat brain tumor cells (10). All cell lines tested
544
activity, whereas
logue of the lysophospholipid,
substances
pos-
2-LPC, the ester ana-
was ineffective (Chart 1 A).
Results of the trypan blue dye exclusion test correlated well
C A N C E R RESEARCH VOL. 43
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Cytotoxic
w i t h r e s u l t s on the i n h i b i t i o n of [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n b y
A L P ( C h a r t 2; T a b l e 2). B o t h i n h i b i t i o n of cell p r o l i f e r a t i o n and
cell d e a t h o c c u r after A L P t r e a t m e n t of the cells. T h e s e r e s u l t s
s u p p o r t p r e v i o u s s t u d i e s , in w h i c h a c y t o t o x i c e f f e c t of A L P
w a s o b s e r v e d w i t h h u m a n l e u k e m i c cells (1, 24), h y p e r n e p h r o m a cells (6), and cells from v a r i o u s u r o l o g i c a l t u m o r s (7).
A f t e r e n z y m a t i c t r e a t m e n t of ET-1 8 - O H w i t h the m i c r o s o m a l
p r e p a r a t i o n c o n t a i n i n g the P t e . H 4 - d e p e n d e n t alkyl c l e a v a g e
e n z y m e , the c y t o t o x i c e f f e c t of the p h o s p h o l i p i d w a s a l m o s t
c o m p l e t e l y r e m o v e d ( C h a r t 3). T u m o r cells, in c o n t r a s t to
normal cells, e x h i b i t m u c h l o w e r levels of alkyl c l e a v a g e a c t i v i t y
(Ref. 1 8, T a b l e 3). T h e s e r e s u l t s e x p l a i n w h y n o r m a l cells t h a t
c a n e f f e c t i v e l y c l e a v e the e t h e r l i n k a g e are less a f f e c t e d b y
A L P (1, 7, 1 4). Also, o n l y the a n a l o g u e s c o n t a i n i n g an O - a l k y l
b o n d e x h i b i t c y t o t o x i c a c t i v i t y w i t h the t u m o r cells. T h e e s t e r
p h o s p h o l i p i d s , e . g . , 2 - L P C , w e r e i n a c t i v e (Refs. 1 a n d 14; see
also C h a r t 1). T h e d i f f e r e n c e in c l e a v a g e e n z y m e a c t i v i t i e s
b e t w e e n t u m o r and normal cells and the f a c t t h a t A L P is a d d e d
in m e d i u m c o n t a i n i n g s e r u m , w h i c h b i n d s A L P , t e n d s to rule
o u t the o c c u r r e n c e of a d i r e c t c y t o l y s i s via a d e t e r g e n t a c t i v i t y
of the l y s o p h o s p h o l i p i d s . It is k n o w n t h a t after e x p o s u r e of
n e o p l a s t i c cells to A L P s e v e r a l vital p a t h w a y s of p h o s p h o l i p i d
m e t a b o l i s m are d i s t u r b e d , w h e r e a s in normal cells t h e s a m e
p a t h w a y s are less a f f e c t e d (1 4). T h e s e e a r l i e r i n d i r e c t o b s e r v a t i o n s led to t h e c o n c l u s i o n t h a t the h i g h c y t o t o x i c i t y of A L P
in n e o p l a s t i c cells is d u e to the i n a b i l i t y of s u c h cells to d e g r a d e
the alkyl e t h e r l i n k a g e (1 4).
In the p r e s e n t s t u d y , the d i r e c t d e m o n s t r a t i o n of the i n v o l v e m e n t of the O - a l k y l b o n d a n d t h e c o r r e s p o n d i n g l o w a c t i v i t y of
the alkyl c l e a v a g e e n z y m e in n e o p l a s t i c cells ( T a b l e 3) w i t h the
a n t i t u m o r a c t i o n of A L P has b e e n d o c u m e n t e d . S u b s t r a t e
s p e c i f i c i t i e s in the b i o c l e a v a g e of t h e O - a l k y l b o n d ( 1 2 , 17)
m i g h t p r o v i d e an e x p l a n a t i o n for the c o m p a r a b l y h i g h c y t o t o x i c
a c t i v i t y of the a n a l o g u e s m o d i f i e d at t h e s n - 2 p o s i t i o n of the
m o l e c u l e (1). T h e O - a l k y l m o i e t y ( s n - 1 ) of c o m p o u n d s s u b s t i t u t e d w i t h f u n c t i o n a l g r o u p s at the s n - 2 p o s i t i o n o t h e r t h a n s n 2 - h y d r o x y l are not s u i t a b l e s u b s t r a t e s for the alkyl c l e a v a g e
e n z y m e (12, 1 7). S i n c e an o b j e c t i v e t u m o r a n d l e u k e m i a res p o n s e has b e e n o b s e r v e d in p a t i e n t s t r e a t e d w i t h ET-1 8 - O C H 3
in a clinical P h a s e I s t u d y (4), the use of A L P - t y p e p h o s p h o l i p i d s
in c a n c e r p a t i e n t s , w i t h d u e c a u t i o n ,
a p p r o a c h to a n t i c a n c e r t h e r a p y .
could
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
provide a new
20.
ACKNOWLEDGMENTS
21.
The authors thank Dr. Gerd Baumann for his helpful discussion and Dr. Tenching Lee and Veronica Fitzgerald for their important contributions to this
investigation.
22.
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FEBRUARY 1983
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545
Cytotoxicity of Alkyl-lysophospholipid Derivatives and
Low-Alkyl-Cleavage Enzyme Activities in Rat Brain Tumor
Cells
Wolfgang E. Berdel, Eva Greiner, Ulrich Fink, et al.
Cancer Res 1983;43:541-545.
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