Vol. 169, No. 3, 1990 June 29, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 959-965
BIOCHEMICAL
GROWTH INHIBITION OF HUMAN FIBROBLASTS BY EPIDERMAL GROWTHFACTOR IN THE PRESENCE OF ARACHIDONIC ACID Takamitsu
Hori, Yoichi Yamanaka, Makio Hayakawa, Shibamoto, Naoto Oku, and Fumiaki Ito
Department of Biochemistry, Setsunan University, Received
May
Faculty Hirakata,
Sayumi
of Pharmaceutical Sciences, Osaka 573-01, Japan
1990
1,
The effect of epidermal growth factor on growth of human fibroblasts was investigated in serum-free medium supplemented with various fatty acids. When linoleic acid, arachidonic acid, or eicosapentaenoic acid was added, each inhibited epidermal growth factor-induced cell growth and showed cytotoxicity at high concentrations (>lO FM). This cytotoxic effect was not observed in the presence of indomethacin, suggesting that prostaglandin production is important in mediation of the growth inhibition. Prostaglandin E3 was increased more than ten thousand times by epidermal growth factor in combination with arachidonic acid. O1990
Academic
Press,
Action cells
of
in
which to
growth
proliferation
is
the acids of
serve
modification phospholipids,
of
and
medium. biological
growth
(1).
such
fatty
of
acid
can medium, as
One
Several
arachidonic cell lines in
as a component the which
responses
factors.
acids
is
acid tissue
cultured group
have
alter
acid
of shown
promote the culture (Z-5).
membrane phospholipids,
known to
arachidonic
of
such
studies
composition achieved by
be
in cultured
Serum contains other substances,
and various
the
fatty
supplementation to the and insulin action (6). acids
has been studied
factors,
including a number of
acids
Fatty
factors
may influence
hormones
substances that fatty
growth
serum-containing
peptide
some of
Fatty
various
grown
nutrients, cells
Inc.
and
of membrane fatty acid insulin
also
serve
precursor for the synthesis of a variety of prostaglandins A number of studies have shown that prostaglandins either
binding as
a
(7). promote
Abbreviations: BSA: bovine serum albumin; DMEM:Dulbecco's modified Eagle's medium; EGF: epidermal growth factor; FCS: fetal calf serum; PGE3:prostaglandin E3; RIA: radioimmunoassay.
959
QOO6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169, No. 3, 1990
(5,8,9)
or inhibit
examined the
the
(10-12)
effect
mitogenic
potent
BIOCHEMICAL
of
action
mitogen
for
cell
fatty
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proliferation.
acids
epidermal
a variety
of
growth cells
oleic acid were neither inhibitory induced growth of human fibroblasts. which itself had no effect on cell combination
with
In this
added to
factor(EGF), (13).
study
serum-free
which
Palmitic
acid
nor stimulatory for However, arachidonic growth,
was
we
medium on
cytotoxic
is
a
and EGFacid, in
EGF. MATERIALS
AND METHODS
Chemicals: Palmitic acid, oleic acid, and linoleic acid were purchased from Serdary Research Laboratories INC.(Ontario, acid (purity; > 99 X) came from Kurita Canada); arachidonic eicosapentaenoic acid (purity; Industries LTD. (Japan); and > 99%) was obtained from Nacalai Tesque, TNC.(Japan). These fatty acids were dissolved in ethanol at 100 mM and stored under N2 gas at -80 OC. Human diferric transferrin, indomethacin, porcine insulin, and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co.(St. Louis, MO). EGF (ultrapure) from mouse submaxillary glands was bought from TOYOBO co .,LTD.(Osaka, Japan); Dulbecco's modified Eagle's medium (DMEM), from Nissui Pharmaceutical Co., LTD.(Tokyo, Japan); and fetal calf serum (FCS), from Filtron PTY, LTD. (Victoria, Australia). Assay of cell growth: Human diploid FS-4 cells, isolated from foreskin tissue, were cultured in DMEM supplemented with 5 % FCS and 60 ug/ml Kanamycin in a CO2 incubator at 37OC. The cells were plated in 35-mm plastrc dishes and cultured for 4 U05) days. The medium of the subconfluent cultures was then changed to DMEM containing 0.2 % FCS and incubation was continued for an additional 2 days. Next, these Gl-arrested FS-4 cells were incubated in DMEM containing 50 rig/ml transferrin, 10 pdml insulin, 2.5 mg/ml BSA, and various fatty acids in the presence or absence of 10 rig/ml EGF. The fatty acids were added to the culture medium after having been mixed with 100 mg/ml BSA. After incubation for the indicated times, the cells were stained with 0.5 % crystal violet as previously described (14). The dye was eluted by addion of 33 % (v/v) acetic acid and the absorbance of the eluate was read at 600 nm on a Shimadzu spectophotometer (UV240). Radioimmunoassay (RIA) analysis: Gl-arrested cells grown in 35-mm dishes were treated with 30 pM fatty acids in the presence or absence of 10 rig/ml EGF. After treatment for 48 h, prostaglandins were extracted from the culture medium by the use of a SEP-PAK Cl8 cartridges (Millipore Co.; Milford, MA).The extracts were assayed for prostaglandin E2(PGE2) with an RIA kit (New England Nuclear; Boston, MA). RESULTS The growth of FS-4 cells was stimulated serum-free medium supplemented with insulin, (Fig.1). This growth stimulatory in the presence of palmitic acid
1.7 fold by transferrin,
EGF in and BSA
action of EGF was not changed or oleic acid. However, when
Vol.
169, No. 3, 1990
BIOCHEMICAL
Palmitic Acid
Control
AND BIOPHYSICAL
Oleic
Linoleic Acid
Acid
RESEARCH COMMUNICATIONS
Arachidonic Acid
EicOSaDentaenoic Acid
Figure 1. Effect of various fatty acids on EGF-induced cell Gl-arrested FS-4 cells were treated with various growth. fatty acids (10 pM) in the presence (striped column) or absence (dotted column) of 10 rig/ml EGF. Cell density was determined on day 4 as described under MATERIALS AND METHODS. Data are expressed as the means + SD of triplicate determinations.
cells
treated
were
eicosapentaenoic density, cell treatment cellular
cells acid,
shape,
rather
than
Since
with
acid,
cytostatic
in nature acid
linoleic
drastically
treatment
(data
not
acid,
decrease by EGF.
of
acid
this
was the
did not inhibited
presence
or eicosapentaenoic that
arachidonic
alone
EGF in the
indicating
arachidonic
thereafter
linoleic
acid , any of which growth was significantly
of
arachidonic
with
or cell The acid,
changed
was
cytotoxic
fatty
acid,
shown).
most inhibitory
we
used this
shows the
fatty acid to study the fatty acid-dependent affect of EGF. Oleic acid, which did not the Figure 2 response to EGF, was also used as a control. effect of concentrations of arachidonic acid and oleic
acid
the
cytotoxic
act
mitogenic
011
presence of dose-dependent even at In
high order
cytotoxicity
ion
mitogenic
response
of
EGF, arachidonic acid inhibited manner; whereas oleic acid
this
to was
in cell
cytotoxicity
to
EGF.
cell had no
In
the
growth in such effect,
a
concentrations. determine
whether the fatty by prostaglandin
mediated
examined the effect of indomethacin, acid-dependent cytotoxicity on fatty indomethacin ( 1 FM- 10 pM that decrease
FS-4 cells
density occurred
to
the
961
production,
we
inhibitor, a cyclooxygenase of EGF. Figure 3 shows )
control
through
acid-dependent
reversed level,
prostaglandin
the EGF-induced indicating that production.
In
Vol.
169,
No.
3, 1990
BIOCHEMICAL
,,
AND
BIOPHYSICAL
I/
RESEARCH
COMMUNICATIONS
I
1
100
10
Fatty Acid (pM) Effect of concentration of exogenous fatty acids Figure 2. treated EGF-induced cell growth. Gl-arrested cells were indicated concentrations of arachidonic acid (0) or oleic 10 rig/ml EGF. Data the presence or absence of (0) in the % increase in cell density over the expressed as untreated control value in the presence of the same concentration fatty acid. Zero percent indicates that EGF-stimulated of growth was reduced to that of EGF-untreated cells. Vertical show standard deviations of the means (n=3).
0.6
001
on with acid are EGFcell bars
-
I
’ -7
I -6
I -5
I -4
lndomethacin IoglMl Figure 3. Effect of indomethacin on arachidonic acid-dependent growth inhibitory action of EGF. Gl-arrested cells were treated with 30 uM arachidonic acid and various concentrations of indomethacin in the presence8(@) or absence (0) of 10 rig/ml EGF. The results are expressed as the mean of duplicate determinations. 962
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. Production of PGE2 in cells treated with the presence or absence of EGF
Fatty
Oleic
fatty
PGE3(ng/106 cells)
Acid 0.387 + ( - 1 0.719 + ( 1.86 479 2 ( 1238
Acid
Arachidonic Acid
0.034
acids
+ EGF
0.295 & 0.029 ( 0.76 ) 0.507 f 0.010 ( 1.31 ) 3647 + 358 ( 9424 )
0.053 ) 125 )
Values are expressed as the means f SD (n=3). Numbers in parentheses indicate the ratio of the experimental t.he control.
the presence of higher demonstrated its mitogenic acid
We next exaJnined and arachidonic
(Table 1). irrespective acid
Oleic of
drastically
potentiated dependent
concentratjons activity.
PGEZ p reduction
of
in cells
acid in the presence had only a small effect
acid
the presence increased
of EGF. In
of the
PGE2 production
prostaglandin
indomethacin, treated
with
EGF oleic
arachidorlic
and this
production
to
or absence of EGF on PGEZ production COJltrast,
by EGF. These results indicate cytotoxic action of EGF is exerted
activation
in
t.hat the through
increase was fatt.y acidEGF-induced
pathway.
DISCC'SSION
FS-4
EGF is
know11 to
cells
(15-17).
stimulate In
growth
this
of
study
various
we exalnined
cells
including
the
effect
of
exogenous fatty acids on this EGF-induced cell growth. The growth of FS-4 cells was inhibited by EGF in combinatjon with linoleic acid, acid
arachidonic and oleic
is known that type
II
acid, acid
or eicosapentaenoic acid; while had no effect on EGF-treated cells.
linoleic
acid
and arachidonic
and
eicosapentaenoic
prostaglandins
type III ones, associated with confirmed
the cytotoxic prostaglandin
by two additional
effect. of production. experimental
acid acid
results:
an inhibitor action
of prostaglandin of EGF; (2) EGF
prostaglandin acid.
production
observed
Cells treated produced PGE2 to
with a level
arachidonic more than 963
the
are
converted
to
is
converted
to
EGF is suggested to be This suggestion was
indomethacin, the cytotoxic
in
palmitic Since it
(1)
addition
of
production, reversed further stimulated
presence
of
acid, but not 1000 t-imes greater
arachidonic with than
EGF, that
Vol.
169,
No.
by control
cells.
treatment
possibility
by
that
higher
than
through
synthase in growth-inhibitory
this
rate
of
growth on
depending
on
We also
previously of
the
FS-4 their
cell
EGF, tumor necrosis produced at sites might
have
Acknowledgment: the Ministry
that
action
absence
of
necrosis of
of the
prostaglandin production
H2 of
stimulatory on the context this
study for
fatty
interaction
we showed FS-4
acids factor
cells, present.
stimulated
acid, but Since the acid (20). metabolites could be of
these
factors
vivo.
was supported by a grant-in-aid Science, and Culture of Japan. REFERENCES
1
Morisaki, N., Lindsey, J.A., Milo, G.E., and Cornwell, D.G.(1983) Lipids 18, 349-352. 2 Gerschenson, L.E., Mead, J.F., Harary, I., and Haggerty, Jr., D.F.(1967) Biochim. Biophys. Acta 131, 42-49. 3 Huttner, J.J., Gwebu, E.T., Panganamala, R.V., Milo,G.E., Cornwell, D.G., Sharma, H.M., and Geer, J.C. (1977) Science 197, 289-291. 4 Bandyopadhyay, G.K., Imagawa, W., Wallace, D.R., and Nandi, S.(1987) J.Biol.Chem. 262, 2750-2756. 5 Skouteris, G.G., ord, M.G., and Stocken, L.A.(1988) J.Cell.Physiol. 135, 516-520. 6 Grunfeld, C., Baird, K.L., and Kahn, C.R.(1981) Biochem.Biophys.Res.Commun. 103, 219-226. 7 Smith, W.L.(1989) Bi0chem.J. 259, 315-324. 8 Nolan, R.D., Danilowicz, R.M., and Eling, T.E.(1988) Mol.Pharmacol. 33, 650-656. 9 Bandyopadhyay, G.K., Imagawa, W., Wallace, D.R., and Nandi, S.(1988) J.Biol.Chem. 263, 7567-7573. 10 Santro, M.G., Phiipott, G.W., and .Jaffe, B.M.(1977) Cancer Res. 37, 3774-3779. 964
and of
arachidonic
the presence of and arachidonate in
increased
indicates
cytotoxic
type
tumor
significance
This study Education,
In
(19). or
and
the in
toxic
induction
show both depending
factor, of inflammation,
biological
of
in
in
alone Another
is
result
synthesis of then increases
growth,
reported growth
by the
increase
for
involves This
factors
concentration cells
an
but insufficient inhibition.
that (18).
stimulates which cells, prostaglandins.
FS-4
suppressed
PGE2 biosynthesis
mechanism
EGF
effects
inhibited
not Thus,
concentration
the other signal molecules present that EGF acts either to be mitogenic
growth
was
COMMUNICATIONS
concentration.
synthase
peptide
inhibitory
RESEARCH
alone.
critical
the a
H2 that
Many
growth
acid
the
cells,
prostaglandin possibility
their
BIOPHYSICAL
production may be necessary acid-dependent growth
amnion
EGF
AND
arachidonic
is
PGE2 is In
However,
with
prostaglandin for the fatty of
BIOCHEMICAL
3, 1990
from
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
11 Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S., and Hayaishi, 0.(1982) Biochem.Biophys.Res.Commun. 105, 956-964. 12 Elias, J.A., Zurier,R.B., Schreiber,A.D., Leff,J.A., and Daniele, R.P.(1985) J.Leukocyte Biol. 37, 15-28. 13 Carpenter, G., and Cohen, S.(1979) Annu.Rev.Biochem. 48, 193-216. 14 Horf! T., Kashiyama, S., Hayakawa, M., Shibamoto, S., Tsujlmoto, M., Oku, N., and Ito, F.(1989) J.Cell.Physiol. 141, 275-280. 15 Sugarman, B.J., Aggarwal, B.B., Hass, P.E., Figari, I.S., Palladino, M.A.,Jr. and Shepard, H.M.(1985)Nature 230,943-945. 16 Vilcek, J., Palombella, V.J., Heriksen-DeStefano, D., Swenson , C., Feinman, R., Hirai, M., and Tsujimoto, M. (1986) J.Exp.Med. 163, 632-643. 17 Hori, T., Kashiyama, S., Oku, N., Hayakawa, M., Shibamoto, S., Tsujimoto, M., Nishihara, T., and Ito, F. (1988) Cell Struct.Funct. 13, 425-433. 18 Casey, M.L., Korte, K., and MacDonald, P.C.(1988) J.Biol.Chem. 263, 7846-7854. 19 Sporn, M.B., and Roberts, A.B.(1988) Nature 332, 217-219. 20 Hori, T., Kashiyama, S., Hayakawa, M., Shibamoto, S., Tsujimoto, M., Oku, N., and Ito, F.(1989) Exp. Cell Res. 185, 41-49.
965
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 959-965
BIOCHEMICAL
GROWTH INHIBITION OF HUMAN FIBROBLASTS BY EPIDERMAL GROWTHFACTOR IN THE PRESENCE OF ARACHIDONIC ACID Takamitsu
Hori, Yoichi Yamanaka, Makio Hayakawa, Shibamoto, Naoto Oku, and Fumiaki Ito
Department of Biochemistry, Setsunan University, Received
May
Faculty Hirakata,
Sayumi
of Pharmaceutical Sciences, Osaka 573-01, Japan
1990
1,
The effect of epidermal growth factor on growth of human fibroblasts was investigated in serum-free medium supplemented with various fatty acids. When linoleic acid, arachidonic acid, or eicosapentaenoic acid was added, each inhibited epidermal growth factor-induced cell growth and showed cytotoxicity at high concentrations (>lO FM). This cytotoxic effect was not observed in the presence of indomethacin, suggesting that prostaglandin production is important in mediation of the growth inhibition. Prostaglandin E3 was increased more than ten thousand times by epidermal growth factor in combination with arachidonic acid. O1990
Academic
Press,
Action cells
of
in
which to
growth
proliferation
is
the acids of
serve
modification phospholipids,
of
and
medium. biological
growth
(1).
such
fatty
of
acid
can medium, as
One
Several
arachidonic cell lines in
as a component the which
responses
factors.
acids
is
acid tissue
cultured group
have
alter
acid
of shown
promote the culture (Z-5).
membrane phospholipids,
known to
arachidonic
of
such
studies
composition achieved by
be
in cultured
Serum contains other substances,
and various
the
fatty
supplementation to the and insulin action (6). acids
has been studied
factors,
including a number of
acids
Fatty
factors
may influence
hormones
substances that fatty
growth
serum-containing
peptide
some of
Fatty
various
grown
nutrients, cells
Inc.
and
of membrane fatty acid insulin
also
serve
precursor for the synthesis of a variety of prostaglandins A number of studies have shown that prostaglandins either
binding as
a
(7). promote
Abbreviations: BSA: bovine serum albumin; DMEM:Dulbecco's modified Eagle's medium; EGF: epidermal growth factor; FCS: fetal calf serum; PGE3:prostaglandin E3; RIA: radioimmunoassay.
959
QOO6-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
169, No. 3, 1990
(5,8,9)
or inhibit
examined the
the
(10-12)
effect
mitogenic
potent
BIOCHEMICAL
of
action
mitogen
for
cell
fatty
of
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
proliferation.
acids
epidermal
a variety
of
growth cells
oleic acid were neither inhibitory induced growth of human fibroblasts. which itself had no effect on cell combination
with
In this
added to
factor(EGF), (13).
study
serum-free
which
Palmitic
acid
nor stimulatory for However, arachidonic growth,
was
we
medium on
cytotoxic
is
a
and EGFacid, in
EGF. MATERIALS
AND METHODS
Chemicals: Palmitic acid, oleic acid, and linoleic acid were purchased from Serdary Research Laboratories INC.(Ontario, acid (purity; > 99 X) came from Kurita Canada); arachidonic eicosapentaenoic acid (purity; Industries LTD. (Japan); and > 99%) was obtained from Nacalai Tesque, TNC.(Japan). These fatty acids were dissolved in ethanol at 100 mM and stored under N2 gas at -80 OC. Human diferric transferrin, indomethacin, porcine insulin, and essentially fatty acid-free bovine serum albumin (BSA) were purchased from Sigma Chemical Co.(St. Louis, MO). EGF (ultrapure) from mouse submaxillary glands was bought from TOYOBO co .,LTD.(Osaka, Japan); Dulbecco's modified Eagle's medium (DMEM), from Nissui Pharmaceutical Co., LTD.(Tokyo, Japan); and fetal calf serum (FCS), from Filtron PTY, LTD. (Victoria, Australia). Assay of cell growth: Human diploid FS-4 cells, isolated from foreskin tissue, were cultured in DMEM supplemented with 5 % FCS and 60 ug/ml Kanamycin in a CO2 incubator at 37OC. The cells were plated in 35-mm plastrc dishes and cultured for 4 U05) days. The medium of the subconfluent cultures was then changed to DMEM containing 0.2 % FCS and incubation was continued for an additional 2 days. Next, these Gl-arrested FS-4 cells were incubated in DMEM containing 50 rig/ml transferrin, 10 pdml insulin, 2.5 mg/ml BSA, and various fatty acids in the presence or absence of 10 rig/ml EGF. The fatty acids were added to the culture medium after having been mixed with 100 mg/ml BSA. After incubation for the indicated times, the cells were stained with 0.5 % crystal violet as previously described (14). The dye was eluted by addion of 33 % (v/v) acetic acid and the absorbance of the eluate was read at 600 nm on a Shimadzu spectophotometer (UV240). Radioimmunoassay (RIA) analysis: Gl-arrested cells grown in 35-mm dishes were treated with 30 pM fatty acids in the presence or absence of 10 rig/ml EGF. After treatment for 48 h, prostaglandins were extracted from the culture medium by the use of a SEP-PAK Cl8 cartridges (Millipore Co.; Milford, MA).The extracts were assayed for prostaglandin E2(PGE2) with an RIA kit (New England Nuclear; Boston, MA). RESULTS The growth of FS-4 cells was stimulated serum-free medium supplemented with insulin, (Fig.1). This growth stimulatory in the presence of palmitic acid
1.7 fold by transferrin,
EGF in and BSA
action of EGF was not changed or oleic acid. However, when
Vol.
169, No. 3, 1990
BIOCHEMICAL
Palmitic Acid
Control
AND BIOPHYSICAL
Oleic
Linoleic Acid
Acid
RESEARCH COMMUNICATIONS
Arachidonic Acid
EicOSaDentaenoic Acid
Figure 1. Effect of various fatty acids on EGF-induced cell Gl-arrested FS-4 cells were treated with various growth. fatty acids (10 pM) in the presence (striped column) or absence (dotted column) of 10 rig/ml EGF. Cell density was determined on day 4 as described under MATERIALS AND METHODS. Data are expressed as the means + SD of triplicate determinations.
cells
treated
were
eicosapentaenoic density, cell treatment cellular
cells acid,
shape,
rather
than
Since
with
acid,
cytostatic
in nature acid
linoleic
drastically
treatment
(data
not
acid,
decrease by EGF.
of
acid
this
was the
did not inhibited
presence
or eicosapentaenoic that
arachidonic
alone
EGF in the
indicating
arachidonic
thereafter
linoleic
acid , any of which growth was significantly
of
arachidonic
with
or cell The acid,
changed
was
cytotoxic
fatty
acid,
shown).
most inhibitory
we
used this
shows the
fatty acid to study the fatty acid-dependent affect of EGF. Oleic acid, which did not the Figure 2 response to EGF, was also used as a control. effect of concentrations of arachidonic acid and oleic
acid
the
cytotoxic
act
mitogenic
011
presence of dose-dependent even at In
high order
cytotoxicity
ion
mitogenic
response
of
EGF, arachidonic acid inhibited manner; whereas oleic acid
this
to was
in cell
cytotoxicity
to
EGF.
cell had no
In
the
growth in such effect,
a
concentrations. determine
whether the fatty by prostaglandin
mediated
examined the effect of indomethacin, acid-dependent cytotoxicity on fatty indomethacin ( 1 FM- 10 pM that decrease
FS-4 cells
density occurred
to
the
961
production,
we
inhibitor, a cyclooxygenase of EGF. Figure 3 shows )
control
through
acid-dependent
reversed level,
prostaglandin
the EGF-induced indicating that production.
In
Vol.
169,
No.
3, 1990
BIOCHEMICAL
,,
AND
BIOPHYSICAL
I/
RESEARCH
COMMUNICATIONS
I
1
100
10
Fatty Acid (pM) Effect of concentration of exogenous fatty acids Figure 2. treated EGF-induced cell growth. Gl-arrested cells were indicated concentrations of arachidonic acid (0) or oleic 10 rig/ml EGF. Data the presence or absence of (0) in the % increase in cell density over the expressed as untreated control value in the presence of the same concentration fatty acid. Zero percent indicates that EGF-stimulated of growth was reduced to that of EGF-untreated cells. Vertical show standard deviations of the means (n=3).
0.6
001
on with acid are EGFcell bars
-
I
’ -7
I -6
I -5
I -4
lndomethacin IoglMl Figure 3. Effect of indomethacin on arachidonic acid-dependent growth inhibitory action of EGF. Gl-arrested cells were treated with 30 uM arachidonic acid and various concentrations of indomethacin in the presence8(@) or absence (0) of 10 rig/ml EGF. The results are expressed as the mean of duplicate determinations. 962
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Table 1. Production of PGE2 in cells treated with the presence or absence of EGF
Fatty
Oleic
fatty
PGE3(ng/106 cells)
Acid 0.387 + ( - 1 0.719 + ( 1.86 479 2 ( 1238
Acid
Arachidonic Acid
0.034
acids
+ EGF
0.295 & 0.029 ( 0.76 ) 0.507 f 0.010 ( 1.31 ) 3647 + 358 ( 9424 )
0.053 ) 125 )
Values are expressed as the means f SD (n=3). Numbers in parentheses indicate the ratio of the experimental t.he control.
the presence of higher demonstrated its mitogenic acid
We next exaJnined and arachidonic
(Table 1). irrespective acid
Oleic of
drastically
potentiated dependent
concentratjons activity.
PGEZ p reduction
of
in cells
acid in the presence had only a small effect
acid
the presence increased
of EGF. In
of the
PGE2 production
prostaglandin
indomethacin, treated
with
EGF oleic
arachidorlic
and this
production
to
or absence of EGF on PGEZ production COJltrast,
by EGF. These results indicate cytotoxic action of EGF is exerted
activation
in
t.hat the through
increase was fatt.y acidEGF-induced
pathway.
DISCC'SSION
FS-4
EGF is
know11 to
cells
(15-17).
stimulate In
growth
this
of
study
various
we exalnined
cells
including
the
effect
of
exogenous fatty acids on this EGF-induced cell growth. The growth of FS-4 cells was inhibited by EGF in combinatjon with linoleic acid, acid
arachidonic and oleic
is known that type
II
acid, acid
or eicosapentaenoic acid; while had no effect on EGF-treated cells.
linoleic
acid
and arachidonic
and
eicosapentaenoic
prostaglandins
type III ones, associated with confirmed
the cytotoxic prostaglandin
by two additional
effect. of production. experimental
acid acid
results:
an inhibitor action
of prostaglandin of EGF; (2) EGF
prostaglandin acid.
production
observed
Cells treated produced PGE2 to
with a level
arachidonic more than 963
the
are
converted
to
is
converted
to
EGF is suggested to be This suggestion was
indomethacin, the cytotoxic
in
palmitic Since it
(1)
addition
of
production, reversed further stimulated
presence
of
acid, but not 1000 t-imes greater
arachidonic with than
EGF, that
Vol.
169,
No.
by control
cells.
treatment
possibility
by
that
higher
than
through
synthase in growth-inhibitory
this
rate
of
growth on
depending
on
We also
previously of
the
FS-4 their
cell
EGF, tumor necrosis produced at sites might
have
Acknowledgment: the Ministry
that
action
absence
of
necrosis of
of the
prostaglandin production
H2 of
stimulatory on the context this
study for
fatty
interaction
we showed FS-4
acids factor
cells, present.
stimulated
acid, but Since the acid (20). metabolites could be of
these
factors
vivo.
was supported by a grant-in-aid Science, and Culture of Japan. REFERENCES
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RESEARCH
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3, 1990
from
Vol.
169, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
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