Download Linoleic Acid Metabolism in Metastatic and

(CANCER RESEARCH 49, 4724-4728. September I, 1989]
Linoleic Acid Metabolism in Metastatic and Nonmetastatic Murine
Mammary Tumor Cells1
Robert S. Chapkin,2 Neil E. Hubbard, Dianne K. Buckman, and Kent L. Erickson
Department of Human Anatomy, School of Medicine, University of California, Davis, California 95616
ABSTRACT
The mechanism(s) by which dietary linoleic acid (18:2n-6) enhances
mammary tumor growth and metastasis is not known. Since arachidonic
acid (20:4n-6)-derived prostaglandins (PC) may play a role in the metastatic dissemination of tumor cells, the ability of two murine mammary
tumor cell lines, 4526 (metastasis positive) and line 168 (spontaneous
metastasis negative), to convert 18:2n-6 into prostaglandins was exam
ined. Cells were initially incubated with |'4qi8:2n-6 and after 8-24 h the
["( '|t;illv acids were quantitated by high-performance liquid chromatography following transesterification. |'4C]18:2n-6 was metabolized primar
ily to |l4C|dihomogammalinolenic acid (20:3n-6) in line 4526 cells and
|"C'|20:4n-6 in line 168 cells. Examination of cellular fatty acid levels
revealed a 20:3n-6/20:4n-6 ratio of 1.79 ±0.36 and 0.20 ±0.02 in line
4526 and 168 cells, respectively. These data are consistent with an
inherently lower A5 desaturase activity in line 4526 relative to 168. To
assess the metabolism of 18:2n-6 into eicosanoid products, the cell lines
were prelabeled with [l4C]18:2n-6 or 0-40 MMnonradiolabeled 18:2n-6
overnight and subsequently stimulated with calcium ionophore A23187
for 1 h. Total PGE production, as determined by radioimmunoassay, was
greater in 168 relative to 4526 cells at all 18:2n-6 concentrations. 14Cprostaglandins detected by high-performance liquid chromatography and
argentation thin-layer chromatography were: PGF,„and PGE, (derived
from 20:3n-6) and PGF2„
and PGE2 (derived from 20:4n-6) from line
4526; PGE, and PGE2 from line 168. PGE,/PGE2 ratios were 1.43 ±
0.07 and 0.23 ±0.03 for 4526 and 168 lines, respectively. Neither cell
line synthesized lipoxygenase products following |'4C]18:2n-6 or [3H]20:4n-6 incubations under the conditions employed. Additional studies
are warranted in order to define the biological properties of 1- and 2series cyclooxygenase products as they relate to tumor cell metastasis.
INTRODUCTION
It is now well established that some human and experimental
tumors are rich sources of prostaglandins derived from arachi
donic acid (20:4n-6) (1,2). The 20:4n-6-derived cyclooxygenase
products have been implicated as modulators of immunoregulation (3,4), tumor promotion (5,6), growth, and dissemination
(7-9). Since the dietary intake of 20:4n-6 is generally low,
tissues are largely dependent upon linoleic acid (18:2n-6), a
shorter chain essential fatty acid which can serve as an initial
unsaturated antecedent for the biosynthesis of 20:4n-6 (10).
Interestingly, in recent dietary studies (11-15), 18:2n-6 has
been shown to enhance the growth and metastasis of rodent
mammary tumors.
The mechanism(s) of enhancement of primary tumor growth
and metastasis by dietary 18:2n-6 is not known. Interestingly,
18:2n-6 effects on tumor growth and dissemination in several
experimental models are blocked by nonsteroidal antiinflamReceived 1/18/89; revised 4/24/89; accepted 5/26/89.
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.
1Supported by NIH Grant CA-47050 and a grant from the National Dairy
Board (administered by the National Dairy Council). Portions of this study were
presented at the joint meeting of the American Society for Biochemistry and
Molecular Biology and the American Society for Cell Biology, January 1989, San
Francisco, California, and the Federation of American Societies for Experimental
Biology, March 1989, New Orleans, Louisiana.
! To whom requests for reprints should be addressed, at Molecular and Cell
Biology Section, Department of Animal Science, Texas A&M University, College
Station, TX 77843-2471.
matory agents, inhibitors of cellular prostaglandin synthesis
(12,13, 16). As a result of these observations and other studies
indicating that 18:2n-6 can be converted into 20:4n-6 by some
mammalian tumors (17, 18), several investigators believe that
20:4n-6-derived eicosanoids play an important role in the pro
motion and metastasis of rodent mammary tumors by dietary
18:2n-6 (4-7, 19). To date, however, few studies have specifi
cally focused on the metabolism of 18:2n-6 in mammary tumor
cells. Therefore, in order to better understand how 18:2n-6 may
affect tumor growth and metastasis, we examined 18:2n-6 me
tabolism in two murine mammary tumor cell lines derived from
a single tumor which developed spontaneously (20, 21). Cell
line 168 is metastatis negative and line 4526 is metastasis
positive (20, 21). In this report, 18:2n-6 uptake, conversion into
20:4n-6 and other polyunsaturated fatty acids, and metabolism
to eicosanoid products were determined. In addition, for com
parative purposes, the profiles of eicosanoid biosynthesis from
20:4n-6 were examined.
MATERIALS AND METHODS
Cell Lines. The two murine mammary tumor cell lines used for these
studies were derived from a single tumor which developed sponta
neously in a BALB/cfC3H female mouse (21, 22). Cell line 168, a clone
from the original tumor, does not spontaneously metastasize (21, 22).
Line 4526 was derived from a lung metastasis of the above original
tumor and is highly metastatic (22).
Cell Culture. Stock cell lines were preserved in liquid nitrogen until
experimental procedures were initiated. After thaw, cells were cultured
for 2 days with 15% heat inactivated calf serum in Eagle's minimum
essential medium with Earle's salts supplemented with 2% essential
vitamin mixture, 1% L-glutamine, 1% non-essential amino acids, 1%
sodium pyruvate and 50 Mg/m' gentamicin at 37°Cin a humidified
atmosphere of 95% air and 5% CO2. Cultures were maintained with
5% calf serum in the supplemented CM.3
Experimental Conditions. Cell lines were grown as described above
to 80% confluency, washed twice with HBSS without calcium or
magnesium, harvested with trypsin-EDTA (0.02-0.05%), resuspended
in CM and transferred to 35- or 60-mm plastic culture dishes or 75cm2 culture flasks. When these cultures had reached 50% confluency,
the medium was removed, the monolayers washed twice with HBSS
with calcium and magnesium, and the medium replaced as described
below for individual experiments.
Conversion of [l4C]Linoleic Acid into Labeled Fatty Acids. Cells pre
pared as described above were incubated in serum-free CM containing
0.025% FAF-BSA and 4.5 MM['4C]18:2n-6 (0.25 MCi/ml) dissolved in
ethanol for 8-24 h. The purity of each lot of [14C]18:2n-6 was assured
upon arrival by HPLC analysis (23). The concentration of ethanol in
the incubation mixture did not exceed 0.1%. Control dishes containing
medium and radiolabel without cells were also incubated and processed
for lipid extraction. Replicate cell monolayers were solubilized in 0.1 N
sodium hydroxide for protein determination using a modified Lowry
method (24) with BSA as standard. After incubation, cells and supernatants were combined and extracted.
3 The abbreviations used are: CM, complete medium; HBSS, Hanks' balanced
salt solution; FAF-BSA, fatty acid free-bovine serum albumin; HPLC, highperformance liquid chromatography; TLC, thin-layer chromatography; PG, pros
taglandin; RT, retention time; RIA, radioimmunoassay.
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LINOLEIC ACID METABOLISM IN MAMMARY TUMOR CELLS
Incubation of Nonradiolabeled Exogenous Linoleic Acid: Tumor Cell
Fatty Acid Composition. The medium of cultures at 50% confluency in
35-mm dishes was replaced with serum-free CM containing FAF-BSA
and 0, 5, 20, or 40 UM nonradiolabeled 18:2n-6 dissolved in ethanol.
Final ethanol concentration did not exceed 0.1 %. After a 24-h incuba
tion the medium was removed, monolayers washed with CM containing
0.1% FAF-BSA for fatty acid determinations, or HBSS with calcium
and magnesium for protein determinations. Cells for fatty acid deter
minations were detached with a rubber policeman and the cellular lipids
extracted using the method of Folch et al. (25). Samples were flushed
with nitrogen and stored at —¿20°C
pending fatty acid analysis.
Prostaglandin Production from [uC|Linoleic Acid and ['111Araelmlonie
Acid. Cell lines were grown in 60-mm culture dishes or 75-cm: flasks
to 50% confluency at which time the medium was replaced with serumfree labeling medium as described above containing 1.8 ¿IM
[I4C]18:
2n-6 (0.1 Ã-Ã-Ci/ml)
or 6.0 nM ['H]20:4n-6 (0.6 /¿Ci/ml).The cells were
incubated with the label for 24 h, the medium removed and the cells
washed with HBSS. The labeled monolayer was then stimulated for 1
h with divalent cation ionophore A23187 (10 ¡IM)
in serum-free CM.
These incubation conditions stimulate maximum prostaglandin pro
duction (data not shown). The viability of 168 and 4526 cells as
measured by trypan blue exclusion was greater than 90%.
Tumor Cell Harvesting and Lipid Extraction. After incubation, cells
and supernatants were collected separately. The cells were washed once
with medium containing 0.1% FAF-BSA and twice with calcium- and
magnesium-free HBSS to remove traces of unincorporated [l4C]18:2n6 or ['H]20:4n-6. Monolayers were then scraped into chloroform/
methanol (2:1, v/v) and extracted by the procedure of Folch et al. (25).
Incubation supernatants were acidified to pH 3 with citric acid and the
lipids were extracted with chloroform/methanol (2:1, v/v). The organic
layer was withdrawn and evaporated to dryness under nitrogen for TLC
and HPLC. Recoveries were determined by counting samples from the
cellular and medium extracts.
Identification of Cell Glycerolipid Metabolites. Aliquots of the cellular
lipid extract were transesterified using 6% methanolic HCI (26). In
incubations with nonradiolabeled 18:2n-6, a known concentration of
heptadecanoic acid (17:0) as an internal standard was added to the total
cellular lipids prior to extraction and transesterification. Fatty acid
methyl esters were partitioned into petroleum ether (b.p. 36-58°C).To
determine the identity of metabolites after incubations, the "(labeled
fatty acid methyl esters were separated by argentation TLC and reversed-phase HPLC. Nonradiolabeled fatty acid methyl esters from
18:2n-6 dosing experiments were quantitated after gas Chromatographie
analysis (26).
Argentation TLC of l4C-labeled Fatty Acid Methyl Esters. Argenta
tion TLC on silica gel G plates impregnated with 10% silver nitrate
was performed as previously described (26), using a solvent system of
diethyl ether/petroleum ether (100:70, v/v) with methyl esters, 18:2n6, 18:3n-6, 20:3n-6, and 20:4n-6 as references. Plates were visualized
using 0.2% 2',7'-dichlorofluorescein in ethanol. The radiolabel migrat
ing with these bands was quantitated using a TLC proportional radio
activity scanner (Berthold, model LB 2832, Wildbad, FRG) equipped
with an Apple lie computer (26).
Reversed-Phase HPLC of "C-labeled Fatty Acid Methyl Esters. The
"( '-labeled fatty acid methyl esters and the appropriate nonradiolabeled
standards were dissolved in acetonitrile and separated on a reversed
phase CIS Ultrasphere ODS column, 5-^m particle size, 4.6 mm x 25
cm (Beckman, Berkeley, CA) using an isocratic solvent mixture of
acetonitrile/water (90:10, v/v). Samples were run for 40 min at a flow
rate of 1.5 ml/min as we have described previously (26). The UV
absorption of the standards in the column effluent was monitored
continuously at 205 nm and the radioactivity quantitated with an on
line radioactivity detector (Radiomatic, Tampa, FL). Counting efficien
cies were determined using [l4C]toluene standards.
Identification of Radiolabeled Soluble Metabolites by Reversed-Phase
HPLC. To ascertain the conversion of [uC]18:2n-6 and ['H]20:4n-6
mobile phase consisted of 66% acetonitrile (0-15 min), 40% acetonitrile
(15-45 min), and 0% acetonitrile (45-55 min). The remainder of the
solvent was buffered with 0.02% phosphorous acid. The solvent flow
rate was 1 ml/min. The column effluent was monitored at 205 nm for
prostaglandins, 235 nm for hydroxy fatty acids, and 280 for leukotrienes. Radioactivity was quantitated using an on-line radioactivity
detector. The radiolabeled metabolites were identified by cochromatography with nonradiolabeled authentic standards; PCF:,, (RT = 13.61
min), PCF,,, (RT = 13.70 min), PGE2 (RT = 17.38 min), and PGE,
(RT = 19.11 min). In addition, to corroborate the identity of the soluble
products, extracts were further evaluated by TLC as described below.
Identification of Radiolabeled Cyclooxygenase Products by TLC. The
radiolabeled soluble products and the appropriate nonradiolabeled
prostaglandin standard were separated on silica gel 60 TLC plates using
the organic phase from ethyl acetate/trimethylpentane/acetic
acid/
water (110:50:20:100, v/v). Plates were visualized using dichlorofluorescein and the radioactivity quantitated with a TLC-proportional
scanner. In addition, 1- and 2-series prostaglandins were further sepa
rated by argentation TLC (27). Selected samples were methylated using
ethereal diazomethane (28) and separated on 10% silver nitrate silica
gel G plates using ethylacetate/acetic acid (99:1, v/v). The Chromato
graphie mobilities of the cyclooxygenase products were: PGF:„(R, =
0.10), PCF,.. (Rr = 0.28), PGE, (R. = 0.37), and PGE, (Rr = 0.48).
Radioimmunoassay of Prostaglandins. Subconfluent cultures of 4526
and 168 cells were incubated with 0, 5, 20, or 40 /IM 18:2n-6 in serumfree CM supplemented with 0.025% FAF-BSA for 16 h. Cultures were
subsequently rinsed and exposed to A23187 (0, 1, or 10 ^M) in serumfree CM for 30 or 60 min. PGE levels of supernatants were determined
using a ['H]PGE2 RIA kit (Advanced Magnetics, Cambridge, MA) that
cross-reacts 50% with PGE, (12).
Reagents and Standards. [l-'4C]9c,12c-Octadecadienoicacid (linoleic
acid, 18:2n-6; 55.6 mCi/mmol, 98% radiochemical purity) and ['H5,6,8,9,1 l,12,14,15]5c,8c,l lc,14c-eicosatetraenoic
acid (arachidonic
acid, 20:4n-6; 100 Ci/mmol, 97% radiochemical purity) were purchased
from New England Nuclear (Boston, MA). All tissue culture medium
was from Whittaker (Walkersville, MD). Calf serum was from Hyclone
(Logan, UT). Silica gel G and silica gel 60 plates were from E. Merck
(Darmstadt, FRG). Fatty acid standards were from Nu Chek Prep
(Elysian, MN) and eicosanoids from Cayman Chemicals (Ann Arbor,
MI) and Dr. J. Rokach (Merck Frosst, Quebec, Canada). Divalent
cation ionophore A23187 was purchased from Sigma Chemical Co.
(St. Louis, MO). ['H]PGE2 RIA kit was purchased from Advanced
Magnetics Inc. (Cambridge, MA).
Statistical Analysis. Data were analyzed using Student's t test (29),
with the upper level of significance chosen at P < 0.05.
RESULTS
Identification and Quantitation of Fatty Acid Metabolites of
Linoleic Acid. Monolayers from lines 168 and 4526 (100-125
ng protein) rapidly incorporated exogenous [MC]18:2n-6 (Fig.
1). The initial rate of uptake (0-4 h) was higher in 4526 versus
168 cells. However, 18:2n-6 incorporation plateaued by 8 h in
100 -|
-C
è
r^l
80 -
x
Û
into eicosanoid products, extracts from the incubation supernatants
were chromatographed using reversed-phase HPLC (27). The soluble
Fig. 1. Percentage uptake of ("C|18:2n-6 (4.5 MM)by line 4526 (•)and 168
metabolites were separated on an Ultrasphere ODS column, 5-iim (D). Cultures contained 2x10" cells. Results are mean ±SEM (n = }) from two
particle size, 4.6 mm x 25 cm, equipped with a guard column. The
separate experiments.
4725
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LINOLEIC ACID METABOLISM IN MAMMARY TUMOR CELLS
both cell lines. Initial characterization of 14C-labeled fatty acid
products by double bond analysis (argentation-TLC) is shown
in Table 1. Relative to line 4526 at 8, 16, and 24 h, line 168
had 3-4-fold higher rates of conversion of [l4C]18:2n-6 into
fatty acid species containing four double bonds (tetraenes).
Additionally, definitive characterization of '4C-labeled fatty
acid products was determined by reversed-phase HPLC. This
data is summarized in Fig. 2, A and B. At all incubation times
examined, line 4526 contained significantly higher (P < 0.05)
[14C]18:2n-6 and [14C]20:3n-6 and significantly lower (P< 0.05)
[IJC]20:4n-6 and [14C]adrenic acid (22:4n-6) levels than line
168. These observations are consistent with argentation-TLC
findings (Table 1), and indicate a possible rate-limiting step at
the level of the AS desaturase (the enzyme that catalyzes the
conversion of 20:3n-6 into 20:4n-6) in line 4526.
Linoleic Acid Dosing: Effect on Tumor Cell Total Lipid Fatty
Acid Composition. Initial examination of cellular total lipid
fatty acid composition prior to the addition of exogenous 18:2n6 revealed that the absolute levels (/ig fatty acid/mg protein) of
20:4n-6 were higher than 20:3n-6 in line 168, and 20:3n-6
higher than 20:4n-6 in line 4526 (Fig. 3, A and B). Cells
incubated in the presence of increasing concentrations of nonradiolabeled 18:2n-6 for 24 h accumulated 18:2n-6. In addition,
at higher substrate concentrations (20 and 40 (¿M
exogenous
18:2n-6), cellular 20:3n-6 in line 4526 and 20:4n-6 in line 168
were significantly elevated (P < 0.05) relative to 0 /^M incuba
tions. These observations suggest that A5 desaturase activity
may be limited in 4526 cells relative to 168 cells.
Metabolism of |'4C|Linoleic Acid into Eicosanoid Products. To
determine the metabolism of 18:2n-6 into eicosanoid products,
cells were prelabeled with [uC]18:2n-6 for 24 h. This prelabeling period was chosen based on earlier experimental results in
which the maximal uptake and conversion of 18:2n-6 into long
chain fatty acid products were observed at 24 h (Figs. 1 and 2).
Cells were subsequently stimulated with divalent cation ionoTable I Characterization of radiolabeled metabolites of linotele acid (I8:2n-fi)
by double-bond analysis
168 and 4526 mammary tumur cell lines were incubated with [ UC"|18:2n-6 (4.5
¡IM)as described in "Materials and Methods." After the combined extraction and
transcstcrification of supernatant and cells, fatty acid methyl esters were separated
according to the degree of unsaturation by argcntation TLC. Results are expressed
as percent distribution of radiolabel, mean ±SEM (n = 3) from two separate
experiments.
Incubation
(h)4526
time
Cells
8
16
24168
Cells
8
1624Number
bondsDienes61.0
±6.9
49.2 ±3.3
3.350.2
55.6 ±
of double
±5.4
40.7 ±2.7
3.028.2
36.9 ±
±1.4
10.0 ±0.6
0.421.8
7.4 ±
phore A23187 for 1 h. This triggering agent is known to
maximally stimulate cellular eicosanoid metabolism in vitro
(30, 31). Preliminary studies indicated maximal eicosanoid
production after 1 h (data not shown). Analysis of incubation
supernatants by reversed-phase HPLC and TLC methodologies
indicated the formation of [14C]prostaglandin products (Table
2). Neither cell line possessed lipoxygenase activity under the
conditions tested. The cyclooxygenase metabolites were iden
tified as PGF,,, and PGE, (derived from [14C]20:3n-6) and
PGF2„and PGE2 (derived from [14C]20:4n-6) from line 4526.
In comparison, only E-series prostaglandins (PGE, and PGE2)
were produced by line 168 cells. In both 4526 and 168 cells,
the generation of 14C-labeled prostaglandins was less than 20%
in prelabeled unstimulated cells (no A23187 added) and
A23187 stimulated cells incubated with cyclooxygenase inhib
itors, ibuprofen (1 and 5 HIM) and indomethacin (10 and 25
¿tM),
relative to A23187 stimulated controls (data not shown).
Interestingly, as shown in Table 2, PGE,/PGE2 ratios were
1.43 ±0.07 and 0.23 ±0.03 for 4526 and 168 lines, respectively.
These studies indicate that in 4526 mammary tumor cells,
18:2n-6 conversion to prostaglandins may be regulated in part
by a rate-limiting A5 desaturase.
Metabolism of ('H)Arachidonic Acid into Eicosanoid Products.
To further examine the mechanisms regulating mammary tu
mor cell eicosanoid metabolism, 168 and 4526 cells were in
cubated with ['H]20:4n-6. Cells were prelabeled and subse
quently stimulated with divalent cation ionophore A23187 as
described above. Line 4526 converted [1H]20:4n-6 into PGE2
and PGF2„,
while only PGE2 was synthesized by line 168 (Table
3). These observations are similar to those made following
incubation with 18:2n-6 (Table 2), where only E-series prosta
glandins were produced by 168 cells. Interestingly, no lipoxy
genase products were detected under the present incubation
conditions using either cell line. In addition, conversion ol | 'I l|
20:4n-6 into cyclooxygenase products was nearly twofold higher
in 168 cells compared to 4526 cells.
Linoleic Acid Dosing: Effect on Tumor Cell Prostaglandin
Production. Quantitative comparison of PGE production by
168 and 4526 cells was further examined using radioimmunoassay (Fig. 4). Consistent with radioactive prostaglandin pro
duction, immunoreactive PGE production was greater in 168
relative to 4526 cells following exposure to 10 UM A23187
stimulus. Similar values were obtained at 1 /<MA23187 (data
not shown). Preincubation with 18:2n-6 had no significant
effect on PGE production.
DISCUSSION
Numerous experiments in animals have demonstrated that
the amount and type of dietary lipid can influence the growth
and dissemination of mammary tumors (13-15, 19). Interest
ingly, recent studies have indicated that 18:2n-6 may be the
±0.3
±2.3
±2.024.4
44.3 ±2.5
±0.8
31.3
1.826.5
±
43.4 ±0.2Trienes32.4 ±0.7Tetraenes6.5
30.0 ±0.8
B aoo-i
Fig. 2. A and H, conversion of [l4C]18:2n-6
(4.5 >iM) to polyunsaluratcd fatty acids. Radiomelabolites were quantilated using revcrsedphasc HP1.C' as described in "Materials and
Methods." Refer to Table 1 for legend details.
* Significantly different. P< 0.05.
16
34
U8:2n-6) J
8
16
34
L (20;2n-6> J
U (2U:3n-ft)J
L (20:4n-6) J
L <22:4n-6)J
4726
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Tally
add
LINOLEIC ACID METABOLISM IN MAMMARY TUMOR CELLS
18:2n-6
Fig. 3. .I. cellular fatty acid concentration
in line 4526 following 24-h incubation with 0.
5, 20, and 40 JIM I8:2n-6. Fatty acids were
quantitated by gas chromatography as de
scribed in "Materials and Methods." Results
represent mean ±SEM (n = 3) from two
separate experiments. * Significantly different
from 0 UM 18:2n-6, P< 0.05. B, cellular fatty
acid concentration in cells from line 168.
18:2n-6
20:3n-6
20:4n-6
10
20
30
0
exogenous 18:2n-6 (uM)
10
exogenous
20
30
40
18:2n-6 niMi
Table 2 Prostaglandin biosynthesis in 168 and 4526 mammary tumor cell lines following ["CJlinoleic acid incubation
Cells were prelabeled with 0.1 ¿iCi/ml[14C]18:2n-6 (1.8 ¡¡M)
for 24 h, washed with HBSS, and subsequently stimulated with A23187 (10 /JM) for 1 h. Supernatants
were extracted and eicosanoids identified and quantitated using combined reversed-phase HPLC and argentation TLC Chromatographie systems as described in
"Materials and Methods." Results represent mean ±SEM (n = 4) from two separate experiments.
Prostaglandin synthesis (dpm/mg protein/h)
Cell lines
4526
1682,445
1Undetectable amounts
PGF2,
±
507a4,364
PCF,,,
±6616,557
PGE2
±600
65,343 ±3,1479.341
Prostaglandin ratios
PGE,
±834
14,792 ±5241.43
PGE,/PGE2
±0.07
0.23 ±0.031.90
PGF,,,/PGF,.,
±0.22
Table 3 Prostaglandin biosynthesis in 168 and 4526 mammary tumor cell lines
following [3H]arachidonic acid incubation
Cells were prelabeled with 0.6 ¿iCi/ml[3H]20:4n-6 (6.0 nvi) for 24 h, washed
with HBSS, and subsequently stimulated with A23187 (10 JIM)for 1 h. Refer to
Table 2 for legend details. Results represent mean ±SEM (n = 4) from two
separate experiments.
This finding is consistent with in vivo total fatty acid composi
tion data of mammary tumor tissue derived from mice injected
with either 168 or 4526 cells (15, 32). Since the dietary intake
of 20:4n-6 is generally low, cells are largely dependent upon
18:2n-6, a shorter chain essential fatty acid which can serve as
Prostaglandin synthesis (dpm/mg protein/h)
an initial unsaturated precursor for the biosynthesis of 20:4n-6
Cell lines
PGE2
PGF2.,
(10). Hence, in metastatic 4526 cells unlike nonmetastatic 168
cells, the membrane levels of 20:4n-6 appear to be restricted by
308,538+ 17,117
4526
272,236 ±14,923
168
894,541 ±6,866
a rate limiting A5 desaturase. This is noteworthy, because
' Undetectable amount.
eicosanoid products derived from 20:4n-6 may play an impor
tant role in the promotion, cell proliferation, and metastatic
12000dissemination of tumor cells (1, 2, 5, 6).
Further examination of the metabolism of 18:2n-6 in mam
•¿5
10000mary tumor cells demonstrated the synthesis of PGF,,,, PGE,
o 8000 (derived from 20:3n-6), PGF2„
and PGE2 (derived from 20:4no.
M 6000 6) in line 4526, and PGE, and PGE2 from line 168 following a
24-h preincubation period. Unlike 4526 cells, 168 cells did not
g
4000 synthesize F-series prostaglandins. These observations were
CU
confirmed following 20:4n-6 incubations, where only E-series
M 2000prostaglandins were produced and suggest that 168 cells lack
9-keto reducÃ-aseactivity (the enzyme which converts E- to FO
10
20
30
40
series prostaglandins). In addition, neither cell line synthesized
exogenous
18:2n-6
detectable lipoxygenase products following 18:2n-6 or 20:4n-6
Fig. 4. Cells were preincubated with 0, 5, 20, or 40 ¿<M
18:2n-6 in the presence
of 0.025% FAF-BSA in serum-free CM for 16 h. Cells were then rinsed with
incubations under the conditions tested. This is significant
HBSS and stimulated with 10 ^M A23187 for 1 h. Supernatants were removed
because
most studies examining the role of dietary fat on
and assayed for PGE by radioimmunoassay as described in "Materials and
Methods." Results are expressed as mean ±SEM (n = 3). A, 168 cells; •¿,
4526
tumorigenesis and immune response have focused only on
cells.
cyclooxygenase derived eicosanoids.
The greater radioactive prostaglandin synthesis by the 168
cell line relative to 4526 is consistent with quantitation of PGE
essential fatty acid primarily responsible for the tumor promot
production by radioimmunoassay. Although both cell lines
ing (13, 14) and metastasis enhancing (15) effects of dietary
produced E-class prostaglandins, differences in PGE, and PGE2
polyunsaturated fat. However, studies specifically examining
tumor cell metabolism of 18:2n-6 are lacking. In the present
production indicate distinct abilities to metabolize polyunsatu
study, we monitored the in vitro metabolism of 18:2n-6 in two rated fatty acid precursors. As shown in Table 2, PGE,/PGE2
ratios were approximately 7-fold higher in 4526 cells relative
murine tumor cell lines derived from a single spontaneous
mammary tumor (20). Our data demonstrate that both cell lines to 168 cells. The ratios are predictable based on the prostaglan
synthesize primarily 18:3n-6, 20:3n-6, and 20:4n-6 fatty acids din fatty acid precursor levels of 20:3n-6 and 20:4n-6. For
from 18:2n-6, indicating the presence of A6 desaturase, elon- example, the 20:3n-6/20:4n-6 cellular fatty acid ratios were
gase, and A3 desaturase activities. Interestingly, AS desaturase
0.20 ±0.02 and 1.79 ±0.36 (derived from Fig. 3) in 168 and
activity (the enzyme that catalyzes the conversion of 20:3n-6 to 4526 cells, respectively. Since cellular fatty acid composition is
20:4n-6) as assessed by the quantitation of cellular fatty acid largely regulated by desaturase enzymes (10), these studies
levels and the metabolism of [14C]18:2n-6 to [14C]20:4n-6 and demonstrate for the first time that mammary tumor cell 18:2n[14C]22:4n-6, is limiting in the highly metastatic cell line 4526.
6 conversion to cyclooxygenase metabolites is regulated in part
4727
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LINOLEIC ACID METABOLISM IN MAMMARY TUMOR CELLS
by a rate-limiting A5 desaturase. Therefore, experiments using
20:4n-6 will not parallel eicosanoid production from 18:2n-6
because direct incubation with 20:4n-6 would bypass this reg
ulatory enzymatic step.
There are numerous reports documenting cyclooxygenase
metabolism of 20:4n-6 in mammary tumor cells (7, 8, 16, 22,
33,34). These studies have focused on the oxidative metabolism
of 20:4n-6, since it is the major fatty acid antecedent of the
biologically active eicosanoids in most cells. However, in the
metastatic mammary tumor cell line 4526, 20:3n-6 is the major
fatty acid cyclooxygenase precursor. Therefore, these cells pri
marily synthesize prostaglandins of the 1-series which are
known to possess antithrombotic properties (35). The tumor
cell production of 1-series prostaglandins could potentially
influence platelet aggregation and thereby alter the association
of the tumor cell with the vessel wall. This is of interest because
platelet aggregation may influence tumor cell metastasis (1,2).
In addition, although the absolute tissue levels of prostaglandins
can be used as a marker of high metastatic potential for neoplastic cells in mammary cancer (8, 22), further studies are
necessary in order to delineate whether qualitative alterations
in the tissue levels of 1- and 2-series prostaglandins are associ
ated with tumor proliferation and metastatic potential.
In conclusion, these studies demonstrate that mammary tu
mor cell 18:2n-6 conversion to prostaglandins is regulated in
part by a rate-limiting A5 desaturase. This finding is significant
in view of the pivotal role that 18:2n-6 plays in promoting
mammary tumorigenesis (11-14). In addition, these studies
demonstrate that highly metastatic (line 4526) and nonmetastatic (line 168) murine mammary cells derived from the same
tumor (20, 21) possess distinct capacities to metabolize 18:2n6 into prostaglandins of the 1- and 2-series. Further studies are
required in order to elucidate the biological properties of 1- and
2-series cyclooxygenase products as they relate to the metastatic
dissemination of tumor cells.
ACKNOWLEDGMENTS
The authors express their thanks to Dr. Vincent Ziboh for use of
HPLC and radiodetection equipment, Craig C. Miller for excellent
technical assistance, and Drs. Amy Fulton and Joshua Rokach for
provision of the tumor cell lines and eicosanoid standards, respectively.
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Linoleic Acid Metabolism in Metastatic and Nonmetastatic
Murine Mammary Tumor Cells
Robert S. Chapkin, Neil E. Hubbard, Dianne K. Buckman, et al.
Cancer Res 1989;49:4724-4728.
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