Download Effects of Some Fatty Acid Esters on the Viability

[CANCER RESEARCH 31, 501-504,
May 1971]
Effects of Some Fatty Acid Esters on the Viability and
Transplantability of Ehrlich Ascites Tumor Cells
Akiko Kato, Kunio Ando, Gakuzo Jamura, and Kei Arima
Laboratory of Microbiology, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan
SUMMARY
Fatty acids, monoglycerides, and some esters of fatty acids
show antitumor activity against Ehrlich ascites tumor in mice.
For investigation of the in vivo mode of action of these lipids,
the effects of those fatty acid esters on animal cells in vitro
were studied in sheep red blood cells and Ehrlich ascites tumor
cells. Throughout these studies, we used fatty acid sucrose
esters because they are soluble in water, so that we could
obtain reproducible results without interference from the
solubility of the samples.
Most of the fatty acids and their esters showed hemolytic
action in sheep red blood cells and tumor cell-killing activity in
vitro. The tumor cells treated with such agents in vitro lost
their transplantability in mice. The sucrose monoester of lauric
acid, which showed the strongest in vivo antitumor activity,
exhibited a strong in vitro activity, but polyoxyethylene
sorbitan monolaurate (Tween 20), which had no antitumor
activity, showed the stronger hemolytic and tumor cell-killing
activities. These results indicate that antitumor activity of
some fatty acid esters cannot be explained only by their
physical attack of the tumor cells.
INTRODUCTION
In our screening for antitumor antibiotics, we found that
fatty acids and monoglycerides isolated from acetone extracts
of some fungal mycelia show antitumor activity in vivo against
Ehrlich ascites tumor in mice (3, 5). Chemical study of the
fatty acids and the monoglycerides indicated that they are the
mixture of the fatty acids or monoglycerides of fatty acids
with carbon numbers of 16 and 18 (2, 6).
Antitumor activity of fatty acids was first reported by
Nakahara (10-13). He reported that olive oil and fatty acids
were effective in increasing the resistance of mice to several
forms of transplantable tumors.
Townsend et al. (21,22) found that lO-hydroxy-2-decenoic
acid from royal jelly suppressed the development of a
transplantable mouse leukemia and the formation of ascitic
tumors in mice. They successively investigated the in vitro
antitumor activity of mono- and dicarboxylic acids and their
hydroxy- and keto- derivatives (9, 17-20), and it was found
that most of the mono- and dicarboxylic acids, when mixed
with 3 different ascites tumor cells at pH values below 5.0 and
prior to inoculation into mice, completely suppressed the
development of the ascites tumor but when acids were mixed
Received February 2, 1970; accepted December 21, 1970.
at higher pH values, only 2-decenoic, linoleic, and linolenic
acids showed the activity.
Unsaturated
fatty acid fraction from the liver of
X-ray-irradiated rabbit has been reported to have in vivo
antitumor activity against the Brown-Pearce sarcoma of rabbit,
Ehrlich ascites tumor in solid form, and several human
carcinomas. The study on the chemical analysis of the fatty
acid fraction was carried out by Seno and Yamamota (16), and
they found that the main components were hexadecanoic,
octadecanoic, octadecenoic, and octadecadienoic acids.
Through the in vivo experiments with Ehrlich ascites tumor
in mice, we studied the antitumor activity of each fatty acid
(1), each monoglyceride (7), and other fatty acid esters (8).
Our results indicated that the antitumor activity of the fatty
acids depended upon the number of the carbon chain and
the degree and the position of unsaturation. It was also found
that some esters were active while others were not. In the
course of these studies, we realized that the insolubility of the
fatty acids and their esters in water might affect the results, or
at least it was a problem to solve in carrying out the
experiments. Tolnai and Morgan (20), meeting with the same
problem, studied the amino acid-fatty acid salts, which were
water soluble, and found that L-lysine and L-arginine laurate
were effective against the Ehrlich and TN3 ascites tumor cells
in vitro. We examined the sucrose ester of fatty acids that were
freely soluble in water.
The antitumor activity of sucrose esters of 7 fatty acids was
studied and reported in a previous paper (8). As summarized in
Table 1, all the sucrose esters inhibit the tumor growth. This
finding of tumor inhibition by fatty acid sucrose esters
enabled us to study the mechanism of action of fatty acids and
their esters on tumor cells.
Most of the works on the activity of fatty acids and their
esters thus far reported were done with water-insoluble
materials. All the works by Townsend et al. (21, 22), Morgan
et al. (9), and Tolnai et al. (17-19) were carried out with
water-insoluble fatty acids themselves, as was the work of
Bennett et al. (4), who studied the effect of fatty acids on
plasma membrane of Ehrlich ascites tumor cells and mouse
erythrocytes.
In these experiments, we used sucrose esters of fatty acids
as in vivo antitumor-active materials, and Tweens as fatty acid
derivatives with water solubility, which are inactive against the
tumor in vivo. Hemolytic activity and in vitro attack of the
tumor cells were studied, and results were compared with in
vivo activity. In the study of the in vitro attack of the tumor
cells, we utilized a differential staining method for dead and
live cells and transplantability assay.
MAY 1971
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501
Akiko Kato, Kunio Ando, Gakuzo Jamura, and Kei Arima
Table I
Antitumor activity of some esters of ¡auricacid against Ehrlich ascites tumor in mice
About 1 million of the tumor cells were implanted i.p. into 5-week-old ddY mice. A solution
or suspension of the samples in 0.86% NaCl solution was administered once daily for 5
successive days. Six mice were used for each group. Control mice were given injections of 0.2
ml of the NaCl solution.
7daysBody
CompoundsMonolaurinMethyl
weight
Tumor
(g)+1.3+4.0+2.0+2.9++
gain
time
(days)>2926>272417182221>30>30>3026>30262528
laurateSorbitan
laurateTween
20Sucrose
+3.7+++
+4.2+++
+6.6+++
+6.9+2.9+
laurateSucrose
1.4+1.8+3.0+1.5+2.9+1.4+1.2+++
myristateSucrose
palmitateSucrose
linoleateControlDose(mg/mouse/day)10.02.520.05.010.05.010.02.516.04.020.05.010.02.518.04.5After
+8.4Survival
Hemolytic
012345678
Activity
Agents with strong antitumor
Ci2-sucrose
Cm-sucrose
Cie-sucrose
C|'e-sucrose
activity
MATERIALS AND METHODS
-I—I—I—I—i—\—I
Sucrose Monoesters of Fatty Acids. These were synthesized
according to the method of Osipow et al. (15). About 3 moles
of sucrose are dissolved in dimethylformamide, and 1 mole of
methyl ester of fatty acid is added with 0.1 mole of potassium
carbonate as an alkaline catalyst. The reaction mixture is
maintained at 90-95° under 80 to 100 mm Hg pressure. After
6 to 9 hr, the solvent is distilled off and the residue is dried in
a vacuum to obtain sucrose ester, monoester being the main
component.
Other Esters of Fatty Acids. These were all available as
chemicals or industrial detergents.
Assay for Antitumor Activity in Vivo. Antitumor activity
was assayed with the use of Ehrlich ascites tumor in mice.
About 1 million of the tumor cells were implanted i.p. in ddY
mice, 5 weeks old, weighing 18 to 22 g. A solution or
suspension of the samples in 0.86% NaCl solution was
administered once daily for 5 successive days, and the tumor
growth and body weight gain after 7 days and life-span up to
30 days were observed. The life-span was shown as the average
life-span of 10 mice in each group.
Assay for Hemolytic Activity. Hemolytic activity of the
esters of fatty acids was assayed with the use of sheep red
blood cells. Sheep red blood cells were collected from the
commercial sheep red blood cell suspension (Toshiba Kagaku,
Tokyo, Japan) by centrifugation at 1500 rpm for 5 min,
washed with 0.86% NaCl solution 3 times, and suspended in
100 volumes of the NaCl solution. Samples were dissolved or
suspended in the 0.86% NaCl solution at the serial dilutions,
and an equal volume of the red cell suspension was added.
After incubation at 37°,for 4 hr, the hemolytic activity was
determined. The activity was expressed by the highest dilution
in which hemolysis was observed visually.
502
1—f-
Cfe-sucrose
C,4-PG
Microbio! MG
Agents with weak antitumor activity
Ciu-MG
Cio-sucrose
dz-FA
Ci2-Me ester
Agents without antitumor
Tween 20
Tween 40
activity
I 2345678
Chart 1. Hemolytic activity of fatty acid esters. Samples were
dissolved or suspended in 0.86% NaCl solution at serial dilutions
starting with 1 mg/ml, and an equal volume of the sheep red blood cell
suspension in the NaCl solution was added. After the incubation at 37°
for 4 hr, the hemolytic activity was determined. The activity was
expressed by the highest dilution in which hemolysis was observed. PC,
propylene glycol; AfC, monoglyceride;FA, fatty acid;A/e, methyl.
Assay for Viability of Tumor Cells. Ascitic tumor cells of
Ehrlich carcinoma maintained by weekly i.p. transplantation
were washed 3 times with PBS1 and suspended at a
1The abbreviation used is: PBS, phosphate-buffered saline.
CANCER
RESEARCH
VOL.
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31
An titumor Activity of Some Fatty Acids
concentration of 1 X IO6 cells/ml. Equal volumes of the the mixture was determined by counting the number of
sample in PBS and the tumor cell suspension were mixed and cells microscopically.
incubated at 37°for 1 hr. The viability of the treated tumor
Assay for Transplantability of Tumor Cells. Ehrlich ascites
cells was determined by staining the cells with safranin dye tumor cells were treated as described for assay for viability
according to the method described by Oda (14). To the except that the tumor cells were suspended at a concentration
of 1 X IO7 cells/ml. After the incubation at 37°for 1 hr, 0.2
reaction mixture, safranin solution in PBS was added to a final
concentration of 0.05%. The percentage of the dead cells in ml of the mixture was implanted i.p. into a ddY mouse. The
survival time and tumor growth of the each mouse were ob
Table 2
served. Ten mice were used for each group, and the control
Effect of some fatty acid esters on Ehrlich ascites tumor cells
mice were given injections of untreated tumor cells.
Figures indicate the percentage of cells stained with safranin. To 0.5
ml of the tumor cell suspension (10* cells/ml), 0.5 ml of the sample
suspension or solution was added. After incubation at 37°for 1 hr,
safranin was added at a final concentration of 0.05%. The percentage of
dead cells was determined by counting the stained cells microscopically.
C, „,C,,, C14, C,0,, and C,28> indicate the esters of capric, lauric,
myristic, stearic, and linoleic acids, respectively.
Percent at
500
Mg/ml
Compounds
Agents
activityC,
with strong antitumor
sucroseC,
-,
esterFungal
4 propylene glycol
monoglycerideAgents
activityC,
with weak antitumor
monoglycerideC,
j
sucroseC,
„
esterAgents
2 methyl
activityC,°8
without antitumor
monoglycerideC,%
esterTween
methyl
20Control100100100100994310010063100
50
Mg/ml
5
Mg/ml
3010052891003
RESULTS
In vivo antitumor activity of some of the fatty acid esters is
shown in Table 1. While monolaurin and sucrose monolaurate
show activity, sorbitan laurate and polyoxyethylene sorbitan
monolaurate (Tween 20) have no activity. Studies on the
activity of other esters (8) showed that most of the sucrose
esters of fatty acids and the propylene glycol ester of myristic
acid were the most effective against the tumor tested and that
Tweens, sorbitan esters, and most of the methyl esters were
ineffective.
Hemolytic activity of some of the fatty acid esters was
studied by the method described in "Materials and Methods."
The purpose of this experiment was to examine whether there
would exist a parallel relationship between hemolytic activity
and in vivo antitumor activity. The result is presented in Chart
1. Hemolytic activity was expressed by the highest dilution in
which hemolysis was observed. As can be seen from the chart,
some of the esters with strong in vivo activity show strong
hemolytic activity while others show very weak activity.
Table 3
Viability and transportability of tumor cells treated
by some esters at 2500 or 500 ng/W cells
Equal volumes of tumor cell suspension (2 X IO7 cells/ml) and the sample suspension or
solution (5 mg/ml or 1 mg/ml) were mixed and incubated at 37°for 1 ht. Viability was
determined by staining the cells with safranin, and transplantability was determined by
implanting 0.2 ml of the incubated mixture into a ddY mouse. Ten mice were used for each
group. The figures after the compounds indicate the final concentration of the compound in
mg/107 cells.
CompoundsNoneLauric
without
cells(%)980880940850670870580102152Mice
time
tumor after
7 days
(%)0100010001000100010001000100808040Survival
(days)18.8>30.020.8>29.920.1>28.422.0>30.020.8>29.3
esterPalmitic
sucrose
esterOleicsucrose
esterLinoleic
sucrose
esterElaidic
sucrose
esterPropylene
sucrose
myristateTween
glycol
20Tween
802.50.52.50.52.50.52.50.52.50.52.50.52.50.52.50.5Viable
MAY 1971
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503
Akiko Kato, Kunio Ando, Gakuzo Tamura, and Kei Arima
Tween 20, which showed no in vivo activity, demonstrated
strong hemolytic activity. Thus there is no relationship
between antitumor activity and hemolytic activity.
Next, we examined in vitro action of those lipids on the
tumor cells, thinking that each lipid might have specific
affinity to the tumor cells and that those with strong affinity
attack the cell and show antitumor activity. In vitro attack of
the cell was measured by viability and transplantability of the
treated tumor cells. The method is described in "Materials and
Methods." In Table 2, viability of the tumor cells treated with
some esters at 3 doses is presented with the reference to their
antitumor activity. Here again, we see no strict relationship
between in vitro and in vivo activities. Table 3 shows the
viability and transplantability of the tumor cells treated with
sucrose and propylene glycol esters of fatty acids and Tweens
at concentrations of 2500 and 500 Mg/107 cells. At the higher
concentration, none of the treated cells were viable; when
inoculated into mice, these cells did not produce tumors.
When the concentration was reduced to 500/ug/107 cells, none
of the sucrose esters and propylene glycol ester showed any
cytotoxic effects on the tumor cells. However, 90% of the cells
treated with Tween 20 at the same concentration, 500/ug/107
cells,
lost
their
transplantability.
viability
and
consequently
4.
5.
6.
7.
8.
9.
their
10.
DISCUSSION
It is significant that fatty acid esters with strong in vivo
activity were less effective in vitro than Tween 20, which did
not show antitumor activity in vivo. Thus antitumor activity
shown by sucrose monoesters and some other esters of fatty
acids cannot be explained only by their physical attack of the
tumor cells. Differences in physiological and biochemical
properties of the esters should differentiate their antitumor
activity. Further studies are under way.
This is the first report in which sucrose esters of fatty acids
are used in biological studies. Our results suggest the
possibility that sucrose esters of fatty acids could be used as
water-soluble derivatives of fatty acids in many experiments.
11.
12.
13.
14.
15.
16.
17.
ACKNOWLEDGMENTS
We are thankful to Mr. Seikichi Suzuki for his kind advice and
discussions.
18.
19.
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CANCER
RESEARCH
VOL.
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31
Effects of Some Fatty Acid Esters on the Viability and
Transplantability of Ehrlich Ascites Tumor Cells
Akiko Kato, Kunio Ando, Gakuzo Tamura, et al.
Cancer Res 1971;31:501-504.
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