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Tumor Biology |
Bassett Research Institute, Cooperstown, New York 13326
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic and eicosapentaenoic acids (EPAs), which differ from LA
and arachidonic acid, respectively, by only a single double bond in the
n-3 position, are recognized cancer chemopreventive and anticachectic
agents. Understanding how this seemingly small structural difference
leads to such remarkable functional differences has been a challenge.
In a previous study, we showed that LA uptake,
[3H]thymidine incorporation into DNA, and total DNA
content were decreased in tissue-isolated hepatoma 7288CTC perfused
in situ with arterial blood containing -linolenic
acid, EPA, or docosahexaenoic acids. The Ki
for the inhibition of LA uptake and [3H]thymidine
incorporation by -linolenic acid was 0.18 and 0.25 mM,
respectively. Here we show that the addition of -linolenic acid or
EPA to arterial blood inhibits tumor FA uptake, including LA, and the
subsequent conversion of LA to the mitogen 13-hydroxyoctadecadienoic
acid (13-HODE) in vivo and during perfusion in
situ. [3H]Thymidine incorporation during
perfusion in situ was also inhibited. Addition of
13-HODE to the arterial blood reversed the inhibition of
[3H]thymidine incorporation but had no effect on FA
uptake. These two n-3 FAs also inhibited FA transport in inguinal fat
pads in vivo and during perfusion in situ
in fed (FA uptake) and fasted (FA release) rats. The effects of EPA and
-linolenic acid on transport of saturated, monounsaturated, and
n-6 polyunsaturated FAs in hepatoma 7288CTC and inguinal fat pads
during perfusion in situ were reversed by the addition
of forskolin (1 µM), pertussis toxin (0.5 µg/ml), or
8-bromo-cyclic AMP (10 µM) to the arterial blood. We
conclude that the antitumor and anticachectic effects of n-3 FAs on
hepatoma 7288CTC and inguinal fat pads in vivo result
from an inhibition of FA transport. These inhibitions are mediated by a
putative n-3 FA receptor via a Gi protein-coupled signal
transduction pathway that decreases intracellular cyclic AMP. A
specific decrease in LA uptake and its conversion to the mitogen
13-HODE causes the tumor growth inhibition. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-linolenic acid (C18:3n3),
EPA (C20:5n3), or docosahexaenoic acid (C22:6n3), inhibited
tumorigenesis (11
, 12) and the growth of rodent tumors
(13
, 14)
and human breast cancer xenografts (15
, 16)
. -Linolenic acid differs from LA by the presence of one
double bond at the n-3 position. EPA, an n-3 FA that is a potent tumor
growth inhibitor, differs from arachidonic acid (C20:4n6) by the n-3
double bond. Dietary arachidonic acid does not stimulate the growth of
rodent tumors in vivo (7
, 8)
. Understanding how
these seemingly small structural differences could lead to such
remarkable functional differences has been a challenge. Research in this laboratory has examined the effects of n-6 and n-3 FAs on the growth of tissue-isolated implants of hepatoma 7288CTC, a transplantable rat tumor. Hepatoma 7288CTC showed a direct relationship between the rates of tumor LA uptake, formation of 13-HODE from LA, and tumor growth in vivo (8) . During perfusion in situ, the incorporation of [3 H]thymidine into tumor DNA was directly proportional to plasma LA concentration and LA uptake (17) and 13-HODE formation (18) . Tumor growth in vivo and [3 H]thymidine incorporation during perfusion in situ were inhibited by a lipoxygenase inhibitor (18) , which blocked 13-HODE formation, and by melatonin, which blocked tumor FA uptake and 13-HODE formation (19) . The effects of the lipoxygenase inhibitor and melatonin were reversed by the addition of 13-HODE to the arterial blood during perfusion in situ. We concluded from these experiments that 13-HODE is the mitogenic agent responsible for LA-dependent growth in hepatoma 7288CTC and suggested that it may have a similar role in the growth of other rodent tumors and in human breast cancer xenografts grown in immunodeficient rodents.
Elucidation of these roles of LA uptake and 13-HODE formation in
hepatoma 7288CTC suggested a new approach to the question of the
interrelationships between n-6 and n-3 FAs and tumor growth. In 1992,
we observed that the addition of -linolenic, EPA, or docosahexaenoic
acid to the arterial blood during perfusion of hepatoma 7288CTC
in situ inhibited tumor LA uptake and the rate of
incorporation of [3
H]thymidine into tumor DNA
(17)
. Uptake of all other saturated, monounsaturated, and
n-6 PUFAs was also inhibited. Ki
values for -linolenic acid were 0.18 mM for LA
uptake and 0.25 mM for
[3
H]thymidine incorporation. There was only a
small uptake of the n-3 FAs by hepatoma 7288CTC. We proposed that
plasma n-3 FAs inhibited tumor growth in vivo and
[3
H]thymidine incorporation during perfusion
in situ by competitively inhibiting LA uptake. However, at
that time, evidence for the presence of specific cellular FATPs for
either n-6 or n-3 FAs in either normal or tumor cells was controversial
(20
, 21) , and, other than the kinetic data, we had no
additional experimental support for this proposal.
In this report, we examine the role of -linolenic acid and EPA on
uptake of plasma FAs and 13-HODE formation in hepatoma 7288CTC in
vivo and during perfusion in situ. The results provide
strong evidence that n-3 FAs inhibit tumor growth by blocking tumor FA
uptake and 13-HODE formation. Similar effects of EPA and -linolenic
acid were observed on FA transport in inguinal fat pads in fasted
tumor-bearing rats in vivo and in fed or fasted normal rats
during perfusion in situ. The evidence presented suggests
that n-3 FAs inhibit uptake of other plasma FAs in tumor and adipose
tissue via a putative n-3 FA receptor-mediated,
Gi protein-coupled signal transduction pathway
that reduces the intracellular cAMP concentration.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hepatoma 7288CTCs were implanted in male Buffalo rats as tissue-isolated tumors as described previously (17, 18, 19 , 22, 23, 24, 25, 26) . Briefly, a 3-mm3 tumor was implanted on the tip of a vascular stalk formed from the superficial epigastric artery and vein. The implant and vascular stalk were enclosed within a parafilm envelope and placed in the inguinal fossa, and the skin incision was closed. Vascularization of the implant was limited to new vessel connections with the epigastric artery and vein, and tumor growth was s.c. The latent period from implantation to first evidence of tumor growth was recorded, and subsequent growth was estimated every 2–3 days from measurements made through the skin (23) . These data were converted to weights, and the growth rates (grams/day) were calculated by linear regression (23) .
A-V Measurements across Hepatoma 7288CTC in Vivo
and during Perfusion in Situ.
Experiments were performed when the estimated tumor weights were 4–6
grams. A-V measurements in vivo were performed between 8 and
10:30 a.m. after a normal nocturnal feeding period (except for the
fasted rats). Procedures for anesthesia, heparinization, surgical
preparation, and maintenance of body temperature of the host rat and
collection of arterial and tumor venous blood samples across the tumor
were performed as described previously (8
, 19
, 22, 23, 24, 25, 26)
.
Blood flow from the tumor vein was 0.11–0.13 ml/min, and blood
was collected passively. Anesthetized host rats were breathing air
unassisted.
Detailed descriptions of the surgical and technical procedures for
perfusion of tissue-isolated hepatoma 7288CTC in situ were
described in previous reports (17, 18, 19
, 24, 25, 26)
. Donor
blood for perfusion (
50 ml) was collected between 8 and 10 a.m.
from either fed or 48-h-fasted Sprague Dawley rats weighing 250–300
grams. Rats were anesthetized with sodium pentobarbital (Abbott
Laboratories, North Chicago, IL; 25 mg/kg body weight; i.p.),
and anticoagulated by i.v. infusion of sodium heparin
(Elkins-Sinn, Inc., Cherry Hill, NJ; 50 units/100 g body weight).
Arterial blood was collected from a carotid catheter, filtered through
cheesecloth, and stored under mineral oil in a stirred plastic
reservoir chilled in ice. This whole-blood perfusate was pumped from
the reservoir through a 37°C water bath and an artificial lung using
a peristaltic pump (Model 1215; Harvard Apparatus, Natick, NA). The pH,
pO2, and pCO2 in samples
collected from the arterial catheter were monitored using a blood gas
analyzer (Model 995; AVL, Graz, Austria) and maintained at 7.4
and 40 and 100 mm Hg, respectively. The vascular connections
(epigastric artery and vein) between tumor and host were severed after
attachment of the tumor to the arterial catheter. The host was
sacrificed, but the hindquarters containing the tumor were monitored
and maintained at 37°C throughout the perfusion. A diagram depicting
the perfusion system is shown in Fig. 1
in Ref. 26
. Calculations of the rates of blood
flow, nutrient uptake, and 13-HODE release were based on actual tumor
weights measured at the end of the experiment.
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-linolenic acid
(plasma concentration, 0.3–0.9 mM) with or
without PTX (0.5 µg/ml), forskolin (1 µM),
8-Br-cAMP (10 µM), or 13-HODE (0.4 ± 0.03 µM). The incorporation of
[3
H]thymidine into tumor DNA was performed by
injecting 20 µl of a solution containing 2 µCi
[methyl-[3
H]]thymidine/gram of estimated
tumor weight into the arterial catheter 20 min before the end of the
perfusion (17, 18, 19
, 25)
. The
[3
H]thymidine made one pass through the tumor
and was washed out during the remaining 20 min of perfusion.
Radioactivity incorporated into tumor DNA was measured by liquid
scintillation using internal standards and is reported as dpm/µg
tumor DNA. DNA was measured fluorometrically in 20% homogenates using
Hoechst dye 33258 (Hoefer Scientific Instruments, San Francisco, CA),
and the procedure was as described previously (28)
. In some experiments, the kinetic effects of n-3 FAs and other agents on tumor FA transport and 13-HODE formation were measured. The 150-min perfusion period was divided into two or three periods, as follows: (a) 66 min after the start of perfusion with untreated donor blood (either fed or fasted rats), n-3 FA was added to the reservoir to give a final plasma concentration of 0.3–0.9 mM, and the perfusion was continued until 150 min; and (b) after a control perfusion for 36 min, the n-3 FA was added, and the perfusion was continued; at 96 min, PTX (0.5 µg/ml), forskolin (1 µM), or 8-Br-cAMP (10 µM) was added to the blood containing n-3 FA, and the perfusion was continued until 150 min. Arterial and tumor venous blood samples were collected every 30 min. At completion of the perfusion, the tumor was removed, weighed, and frozen at -20°C. Whole blood samples were centrifuged for 10 min at 10,000 x g (4°C), and plasma was collected and frozen at -20°C.
A-V Measurements across Inguinal Fat Pads in Vivo
and during Perfusion in Situ.
The procedures described above for A-V blood collections across
tissue-isolated tumors were modified for A-V measurements across the
inguinal fat pad. The in vivo measurements were made across
a tumor and inguinal fat pad simultaneously in the same tumor-bearing
rat. The host rat was anesthetized and heparinized, and the tumor (on
the animal’s left side) was prepared for A-V measurement as described
above. The right inguinal fat pad was prepared as follows: the
epigastric vessels supplying the caudal pole of the fat pad were
exposed by a 3-cm incision in the right inguinal fossa. A butterfly
infusion catheter (number 4573; Abbot Hospital Products, North Chicago,
IL) was inserted into the vein, draining the fat pad. Venous blood was
allowed to flow passively (the venous blood flow rates from the fat
pads were about 80 µl/min). Blood samples (0.5 ml) were collected
simultaneously from the fat pad and tumor veins. An arterial blood
sample was collected from the catheter in the carotid artery midway
through the venous sample collections. EPA (sodium salt in saline)
estimated to give a plasma concentration of 0.3–0.5
mM in a 250- to 300-gram rat with a whole blood
volume of 10 ml was infused into a catheter in the jugular vein. A
second set of blood samples was collected 1–2 min later. At the end of
the experiment, the fat pad and tumor were excised, weighed, and frozen
at -20°C.
The procedure used for perfusion of the left inguinal fat pad in situ in non-tumor-bearing rats was as follows. The caudal epigastric artery and vein supplying the fat pad were exposed, and the vein was cannulated as described above. The catheter carrying the warmed, oxygenated donor blood from the reservoir was inserted into the femoral-epigastric arterial trunk leading to the fat pad. The pump settings were adjusted to provide a flow from the venous catheter of about 80 µl/min. When a uniform venous flow rate was established, the femoral artery and vein distal to the fat pad were ligated, and the host was exsanguinated through the carotid catheter. Death of the host did not affect the venous flow from the fat pad. Arterial and venous blood samples were collected at 30-min intervals, and treatments with n-3 FAs, PTX, forskolin, and 8-Br-cAMP were as described above. A-V measurements in vivo and during perfusion in situ were performed between 7:30 a.m. and 1 p.m. The surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee.
Lipid Extraction and Analysis.
Total plasma lipids were extracted from 0.2 ml of arterial or venous
plasma after the addition of internal standards (8
, 17, 18, 19
, 24
, 25)
. Duplicate extracts for FA analysis were saponified,
methylated, and assayed by GC. In some experiments, the arterial and
venous plasma samples (containing four added internal standards) were
extracted; separated by TLC into FFA, TAG, PL, and CE lipid fractions;
eluted from the plates; saponified; and methylated; and the FA contents
were analyzed by GC (24)
. 13-HODE was measured by
high-performance liquid chromatography as described previously
(8
, 18
, 19)
. A-V measurements were converted to rates of
FA uptake or release and expressed as micrograms or
micromoles/minute/gram fat pad or tumor. 13-HODE
release was expressed as nanograms/minute/gram tumor. Unless otherwise
indicated, total FA represents the sum of the seven major plasma FAs
[myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic
acid, LA, and arachidonic acid (and EPA, when present)] in rat blood
plasma. Plasma concentrations for total FA, LA, EPA, and -linolenic
acid are given as micromolar ± 1 SD, unless otherwise
indicated.
Glycerol Analysis.
Glycerol concentrations were measured spectrophotometrically using an
Ultraspec 4000 UV/visible spectrophotometer equipped with a Compaq
Deskpro 2000 computer (Pharmacia Biotech, Ltd., Piscataway, NJ).
Perchloric acid extracts of plasma (22
, 26)
derived from
the arterial and venous blood collections were analyzed using a kit
purchased from Sigma/Aldrich (St. Louis, MO). Glycerol release from the
fat pads is reported as micrograms/minute/gram fat pad.
Statistical Analysis.
Results are expressed as mean ± SD and were compared
using one-way ANOVA followed by a Student-Newmann-Keuls
multiple-comparison test (29)
. Differences among the
groups were considered statistically significant at
P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Negative values (more FAs in venous blood than in arterial blood)
represent FA release; positive values represent FA uptake. The rate of
FA uptake by the tumor was nearly double the rate of FA release from
the right inguinal fat pad. In the fasted tumor-bearing rat, relative
to the fed rat, the plasma concentration of LA (8)
and the
rates of tumor 13-HODE formation and growth were increased (8
, 18)
. After collection of the first set of arterial blood samples
and two venous blood samples, EPA (sodium salt, in saline) was injected
into the host via the jugular vein catheter; the second set of blood
samples was collected 1 min later. Total collection time was about 10
min. The presence of EPA in the arterial blood inhibited the uptake of
plasma FAs in the tumor, except for that of EPA itself, and completely
inhibited FA release by the inguinal fat pad. The rate of total FA
uptake by hepatoma 7288CTCs after EPA injection was accounted for by
the uptake of EPA; the values for total FA and EPA uptakes were not
different (P > 0.05). As a result of the
block in LA uptake, tumor release of 13-HODE was inhibited. 13-HODE is
undetectable in rat arterial blood plasma (8)
. Fat pads
did not release 13-HODE into the venous blood.
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.
Steady-state rates of total FA and LA uptake and 13-HODE release were
evident during the first 30 min of perfusion. The endogenous EPA
concentration in the donor arterial blood was 
40 µM,
and uptake was too small to be measured accurately. The addition of EPA
to the arterial blood at 36 min promoted an EPA uptake of
1.41 ± 0.15 µg/min/g tumor and inhibited the uptake
of total plasma FAs, including LA. In the absence of LA uptake, release
of 13-HODE stopped. As shown in Table 1
, the rate of FA uptake by
hepatoma 7288CTC following the addition of EPA was accounted for by the
uptake of EPA itself. The addition of 8-Br-cAMP to the perfusate
returned the rates of tumor total FA and LA uptakes and 13-HODE release
to pre-EPA rates. The stimulation by 8-Br-cAMP of the uptake of
plasma-saturated, monounsaturated, and n-6 PUFAs occurred, despite the
presence of EPA in the perfusate. In separate experiments (data
not shown), we found that -linolenic acid (0.21 mM
plasma concentration) was as effective as EPA in blocking FA uptake and
13-HODE release; the action of -linolenic acid was also reversed by
8-Br-cAMP. PTX and forskolin were as effective as 8-Br-cAMP in
reversing the inhibition of FA transport by either EPA or -linolenic
acid.
Steady-State Rates of FA Uptake, 13-HODE Release, and
[3
H]Thymidine Incorporation in Hepatoma 7288CTC Perfused
in Situ: The Effect of EPA and EPA plus Forskolin,
8-Br-cAMP, PTX, or 13-HODE.
In these experiments, 18 tumor-bearing rats were divided into a
control group and five treatment groups (three rats/group). The rats in
the six groups and the blood donor rats were fasted for 48 h
before the start of the experiments. Mean plasma concentrations for the
major FAs in fasted donor arterial blood (six batches, 50 ml each) were
as follows: (a) myristic acid, 20 ± 5
µM; (b) palmitic acid,
1.28 ± 0.06 mM; (c)
palmitoleic acid, 0.13 ± 0.03 mM;
(d) stearic acid, 0.83 ± 0.04
mM; (e) oleic acid, 1.06 ± 0.14 mM; (f) LA,
1.59 ± 0.14 mM; (g)
arachidonic acid, 1.93 ± 0.17 mM;
and (h) EPA, 30 ± 10
µM. A-V measurements across tumors in the
control group represented the baseline steady-state rates for FA
uptake, 13-HODE release, and [3
H]thymidine
incorporation. Arterial blood perfusates for the five treatment groups
also contained added EPA, EPA + 1 µM
forskolin, EPA + 10 µM 8-Br-cAMP,
EPA + 0.5 µg/ml PTX, or EPA + 0.40 ± 0.02 µM 13-HODE, respectively. The
final mean plasma EPA concentration in the arterial blood used for
perfusion of the five treatment groups was 0.52 ± 0.09
mM. The 13-HODE concentration added to the
arterial blood was calculated to equal or exceed the concentrations
observed in tumor venous blood plasma in fasted rats.
Comparisons of the steady-state rates of total FA and LA uptakes by
tumors in the control and treated groups are shown in Fig. 2, A and B
, respectively. The addition of EPA
caused a complete inhibition of total FA and LA uptake. These
inhibitions attributable to EPA were reversed in the presence of either
forskolin, 8-Br-cAMP, or PTX. The addition of EPA + 13-HODE
did not reverse the inhibited uptake of FA. EPA uptake in the control
tumor group was too low to be measured accurately. However, EPA uptake
was observed in the EPA treatment groups as follows: (a)
EPA, 0.40 ± 0.09 µg/min/g tumor; (b)
EPA + forskolin, 1.02 ± 0.07 µg/min/g
tumor; (c) EPA + 8-Br-cAMP, 0.98 ± 0.05 µg/min/g tumor; (d) EPA + PTX,
1.70 ± 0.13 µg/min/g tumor; and (e)
EPA + 13-HODE, 0.42 ± 0.03 µg/min/g tumor.
Each value represents 16 measurements for each treatment group.
Rates of EPA uptake were low compared with the uptake of either total
FA or LA (Fig. 2)
. However, analysis (ANOVA) of these rates, normalized
to a constant rate of EPA supply to the tumor, indicated that
forskolin, 8-Br-cAMP, and PTX significantly increased EPA uptake
(P < 0.05) relative to that in the
EPA-treated and EPA + 13-HODE-treated groups.
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. The addition of either forskolin, 8-Br-cAMP, or PTX,
which reversed the EPA inhibition of LA uptake, restored tumor 13-HODE
release to control rates. Thus, EPA did not directly affect the
enzymatic generation of 13-HODE from LA. Also, despite the presence of
EPA, hepatoma 7288CTC removed significant amounts of 13-HODE from the
arterial blood in the EPA + 13-HODE-treated group. Fig. 3B
shows the rate of [3
H]thymidine
incorporation that occurred in control tumors. LA-dependent synthesis
and release of 13-HODE supported [3
H]thymidine
incorporation (18)
. The presence of EPA abolished 13-HODE
release (Fig. 3A)
and [3
H]thymidine
incorporation into hepatoma 7288CTC (Fig. 3B)
. This
inhibition was reversed by the presence of forskolin, 8-Br-cAMP, or
PTX. The addition of 13-HODE restored
[3
H]thymidine incorporation, although EPA was
present, and LA uptake was inhibited (Fig. 2B)
.
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Fig. 4A
shows that release of FFAs and glycerol from inguinal fat
pads in fasted rats became inhibited when EPA was added 66 min
after the start of the perfusion. The inhibited rates of FFA and
glycerol release were completely restored to pre-EPA rates after
the addition of 8-Br-cAMP to the arterial blood (Fig. 4B)
.
Although trace amounts of EPA may have entered the fat pads, there was
no measurable EPA uptake. In Fig. 4, A and B
, the
molar ratio of FA release:glycerol release was 0.5–0.6,
suggesting that FA reesterification occurred in the inguinal fat pads.
Although an internal standard (pentadecanoic acid) was added to correct
for FFA losses during extraction and chromatography, and antioxidants
were added to the TLC solvent system, PUFAs are subject to loss by
autooxidation during TLC. Glycerol may also have been produced in
excess.
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shows the rates of total FA uptake and glycerol release in inguinal fat
pads in fed rats during perfusion in situ. Glycerol was
released during periods of net FA uptake. The addition of EPA to the
arterial blood 36 min after start of the perfusion inhibited uptake of
FAs from the arterial blood and glycerol release (Fig. 5A)
.
Despite the inhibition of plasma saturated, monounsaturated, and
n-6 PUFA uptake, low but measurable rates of EPA uptake persisted. The
addition of PTX restored uptake of the saturated, monounsaturated, and
n-6 PUFAs but did not affect EPA uptake significantly (Fig. 5B)
. PTX, 8-Br-cAMP, and forskolin (data not shown) were
equally effective in reversing the inhibition of FFA release or uptake
caused by EPA or -linolenic acid.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a recent report (19) , we showed that the neurohormone melatonin inhibited the uptake of saturated, monounsaturated, and n-6 PUFAs and the formation of 13-HODE in hepatoma 7288CTC via a Gi protein-coupled signal transduction pathway. Melatonin acts through cell surface receptors to decrease the intracellular concentration of cAMP (32) . The actions of melatonin were reversed by PTX, forskolin, 8-Br-cAMP, and the Servier compound S20928, a melatonin receptor antagonist (33) . Compound S20928 had no effect on the inhibition of FA uptake by n-3 FAs (data not shown), indicating that despite the shared postreceptor pathways, melatonin and n-3 FAs are likely to have different receptors. We propose that the decreased LA uptake and 13-HODE release caused by n-3 FAs in hepatoma 7288CTC act via a putative n-3 FA receptor-mediated, Gi protein-coupled signal transduction pathway that decreases intracellular cAMP. This mechanism may explain why LA-dependent growth of many rodent tumors (3 , 4 , 12) and human breast (9 , 10 , 16 , 34 , 35) and prostate (36) cancer xenografts in immunodeficient rodents is inhibited by n-3 FAs. Preliminary experiments indicate that 13-HODE release and [3 H]thymidine incorporation in MCF-7 human breast cancer xenografts in nude rats perfused in situ are inhibited by melatonin and EPA and that the addition of 13-HODE to the arterial blood restored [3 H]thymidine incorporation but not LA uptake.4 Although 13-HODE is a mitogen in rat hepatoma 7288CTC and in MCF-7 human breast cancer xenografts, it may not be mitogenic in all LA-requiring tumors. There is evidence that 13-HODE, which is formed in normal human colon epithelium (37) and guinea pig epidermis (38) , has antiproliferative effects in colon carcinomas (37) and hyperproliferating epidermis (38) . It seems that the LA-derived mitogen in neoplastic cells will prove to be tissue specific. It is important to note, however, that n-3 FAs and melatonin blocked the uptake of both plasma LA and arachidonic acid. Therefore, tumor growth that is dependent on a lipid mediator derived from either plasma LA or arachidonic acid will be inhibited.
Other mechanisms were proposed to explain the antitumor actions of n-3
FAs. Competitions between n-3 FAs and LA and arachidonic acid for the
enzymes of elongation, desaturation, and arachidonic acid metabolism
were proposed to slow tumor growth by decreasing production of
growth-enhancing lipid mediators (13
, 39
, 40)
. This
mechanism does not seem to operate in hepatoma 7288CTC. First,
arachidonic acid itself did not stimulate growth in rodent tumors
(7)
, even in essential FA-deficient rats (8
, 17)
. Second, the growth-inhibitory effects of n-3 FAs were
reversed by the addition of forskolin, PTX, and 8-Br-cAMP (Fig. 1)
. n-3
FAs had no effect on 13-HODE formation in the presence of these agents,
indicating that the proposed competitions between LA, -linolenic
acid, and EPA did not adversely affect lipoxygenase activity. Also
proposed was a decrease in tumor promotion caused by changes in PL
n-3:n-6 FA ratios (40)
and an increase in the potential
for auto-oxidation caused by incorporation of n-3 FAs into tumor
membrane lipids (10
, 34)
. We have no evidence that our
results were influenced by these reactions. However, they may become
important as tumor growth progresses.
The effects of n-3 FAs on FA transport in inguinal fat pads were
particularly interesting. In fasted rats, n-3 FAs decreased the rate of
release of FFAs and glycerol originating from lipolysis of TAGs to
essentially zero (Fig. 4A)
. The release was restored by
8-Br-cAMP (Fig. 4B)
and by PTX or forskolin (data not
shown). This finding confirmed the report of Tisdale and Beck
(41)
, who showed that EPA inhibited lipolysis (as measured
by glycerol release) and lowered the intracellular cAMP concentration
in isolated murine epididymal adipocytes in vitro. In their
experiments, the effects of EPA were reversed by PTX. Tisdale
(42)
and Price and Tisdale (43)
suggested
that EPA acted through a putative EPA receptor-mediated,
Gi protein-coupled pathway. It is unclear at this
point whether added 8-Br-cAMP restored FFA and glycerol release from
the fat pads by increasing lipase activity, FFA transport, or both. A-V
measurements performed across intact inguinal fat pads in
vivo and during perfusion in situ are unable to
distinguish between these mechanisms. Hormone-sensitive lipase, the
enzyme believed to be responsible for lipolysis in adipose tissue, is
activated by phosphorylation by protein kinase A. In a recent report,
Osuga et al. (44)
showed that when both alleles
for the hormone-sensitive lipase were disrupted in knockout mice,
adipocytes in white adipose tissue were enlarged, but the mice were not
obese. White adipose tissue still retained 40% of the TAG lipase
activity compared with wild-type unaltered mice. Thus, one or more
additional lipases with unknown properties may also contribute to
lipolysis in adipocytes.
In fed rats (Fig. 5)
, uptake of the saturated, monounsaturated, and n-6
PUFAs was inhibited by n-3 FAs in a manner similar to that observed in
hepatoma 7288CTC. Utilization of plasma FAs and glucose by inguinal fat
pads and their metabolism and storage as TAGs are anabolic reactions
mediated by feeding, insulin, and other agents. Intracellular cAMP
concentrations are at basal levels in adipose tissue during TAG
synthesis (45)
. This basal level was sufficient to support
substantial rates of total FA uptake and a small glycerol release. EPA
reduced these rates to about zero, but the pre-EPA rates were restored
by 8-Br-cAMP and forskolin. The quantitative relationships between
intracellular cAMP concentrations, FFA transport, lipolysis, and other
cAMP-regulated reactions are not yet fully understood
(45)
. Presumably, basal levels of cAMP support uptake of
the saturated, monounsaturated, and n-6 PUFAs directly by an undefined
mechanism; FFA release may require higher intracellular cAMP
concentrations. Experiments to determine the effects of n-3 FAs, PTX,
and forskolin on the basal levels of cAMP in hepatoma 7288CTC and
inguinal fat pads in vivo and during perfusion in
situ are in progress. We suggest that control by n-3 FAs of FFA
uptake and release in inguinal fat pads is the likely mechanism for
their anticachectic actions to preserve host fat stores in
tumor-bearing animals (41, 42, 43)
.
n-3 FAs do not seem to compete directly with the
plasma-saturated, monounsaturated, and n-6 PUFAs for entry into
hepatoma 7288CTC, as we suggested previously (17)
. Rather,
the evidence indicates that the uptakes of the n-3 FAs and other plasma
FAs occur independently of each other. At the plasma n-3 FA
concentrations used in these experiments, uptake of the saturated,
monounsaturated, and n-6 PUFAs was completely inhibited. Despite this
inhibition, n-3 FA uptake continued at measurable rates in hepatoma
7288CTC and inguinal fat pads in fed rats and was less affected by the
addition of PTX, forskolin, or 8-Br-cAMP, agents that completely
restored uptake of the plasma saturated, monounsaturated, or n-6 PUFAs.
Previous experiments showed that the uptake of FAs was directly related
to the rate of supply to hepatoma 7288CTC (8
, 24)
and that
the inhibitory the effects of the n-3 FAs were dose dependent
(17)
. The plasma Ki value
for inhibition of LA uptake by -linolenic acid was 0.18
mM, a value that is well within the range of
concentrations observed in rodents fed dietary n-3 FAs (11
, 14
, 46
, 47)
. Therefore, at a given n-3 FA concentration, an increase
in the plasma LA concentration will attenuate the negative effect of
the n-3 FA. Hudson et al. (14)
described this
interaction between ingested LA and EPA in mice bearing the MAC16 colon
adenocarcinoma. Groups of mice fed laboratory chow were given daily
oral doses (by gavage) of water, LA, EPA, or EPA + LA. EPA
administration increased the serum EPA concentration and reduced the
tumor growth rate relative to that of the water- and LA-treated mice.
Treatment with EPA + LA increased the plasma LA concentration
and the tumor growth rate but did not change the serum EPA
concentration.
Understanding the mechanism by which FAs are transferred from the
plasma to the cell interior is a critical issue in cancer growth,
cachexia, and lipid homeostasis. The mechanism is not yet clearly
resolved. Experimental evidence supports two hypotheses: (a)
that the lipophilic FAs penetrate cell membranes by diffusion (reviewed
in Ref. 20
); and (b) that transport requires
specific carriers (reviewed in Ref. 21
). Recent findings
have strengthened the second hypothesis that specific transporters with
high affinity for long-chain FAs are involved. FATP1, a murine membrane
protein, was shown to facilitate FA uptake in 3T3-L1 adipocytes
(48)
. The FATP1 gene is a member of a
family of five to six related genes represented among several tissues
within a species and with homologues in different species
(49)
. FATP1 mRNA is highly expressed in tissues with high
rates of FA metabolism, such as the heart, adipose tissue, skeletal
muscle, liver, testis, and intestine (48, 49, 50)
. Relative to
normal liver, FATP mRNA seems to be overexpressed in hepatoma 7288CTC
(19)
, suggesting that its expression may be linked to
progression. The upstream region of the murine FATP1 gene
contains an insulin response element (51)
, and expression
of FATP1 mRNA levels in 3T3-L1 adipocytes is up-regulated by nutrient
depletion and down-regulated by insulin (52)
. A peroxisome
proliferator-activated response element was identified in the murine
FATP1 gene (53)
. LA, which is a natural ligand
for peroxisome proliferator-activated receptors 
and ,
up-regulated FATP1 expression (53)
. The primary sequence
of FATP1 has similarities to that of a large family of
acyl-CoA-transferases, and evidence was presented that FATP1 has
long-chain FA acyl-CoA transferase activity; FA influx may be coupled
to esterification (54)
. It is not yet known whether FA
efflux, such as that which occurs during lipolysis, requires FATP1, or
whether efflux occurs by diffusion. Given the critical role of cellular
FA transport in energy metabolism and lipid homeostasis, it seems very
likely that additional controls of FATP1 gene transcription
will be found. In this study and in a previous report we have shown
that n-3 FAs and melatonin (19)
exert important kinetic
controls over FA uptake in hepatoma 7288CTC and over FA uptake and
release in inguinal fat pads in vivo and during perfusion
in situ. These controls are mediated by well-documented
(melatonin) and putative (n-3 FAs) cell surface receptors. The
information is transmitted inside the cells via
Gi protein-coupled signal transduction pathways
and is responsible for the antitumor and anticachectic properties of
these agents.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 A preliminary report of this research was
presented at the 2000 AACR Annual Meeting in San Francisco, California
on April 4, 2000. The research was supported by National Cancer
Institute Grant R01 CA76197 (to D. E. B.) and the Stephen C. Clark
Research Fund of the Mary Imogene Bassett Hospital. L. A. S.,
R. T. D., and D. E. B. contributed equally to this research. 
2 To whom requests for reprints should be
addressed, at Bassett Research Institute, One Atwell Road, Cooperstown,
NY 13326. Phone/Fax: (406) 777-4360; E-mail: lensauer{at}juno.com
3 The abbreviations used are: LA, linoleic acid;
FA, fatty acid; PUFA, polyunsaturated FA; 13-HODE,
13-hydroxyoctadecadienoic acid; EPA, eicosapentaenoic acid; A-V,
arteriovenous difference; FFA, free fatty acid; TAG, triacyglycerol;
PL, phospholipid; CE, cholesterol ester; PTX, pertussis toxin; cAMP,
cyclic AMP; 8-Br-cAMP, 8-bromo-cyclic AMP; FATP, fatty acid transport
protein; GC, gas chromatography.
Received 3/20/00. Accepted 7/20/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-3 and -6 fatty acids on initiation and postinitiation stages of colon carcinogenesis. Cancer Res., 51: 487-491, 1991.-3 fatty acids on growth of a rat mammary tumor. J. Natl. Cancer Inst., 73: 457-461, 1984.
-3 fatty acids on human breast cancer growth and metastasis in nude mice. J. Natl. Cancer Inst., 85: 1743-1747, 1993.-6 and -3 fatty acids on [3H]thymidine incorporation in hepatoma 7288CTC perfused in situ. Br. J. Cancer, 66: 297-303, 1992.[Medline]
-linolenic acid on colon carcinogenesis in rats. Jpn. J. Cancer Res., 82: 1089-1096, 1991.[Medline]
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in A
and B) was <5 µM. The mean arterial blood
EPA concentration in A (added at 66 min) and
B (added at 36 min) was 0.78 ± 0.17
mM. Negative values represent FFA and glycerol release from
the fat pads. There was no uptake of the added EPA. Mean weight was
5.0 ± 0.3 g (n = 6).
in A) was <20
µM. The mean arterial blood plasma:EPA concentration in
A (added at 66 min) and in B (added at 36
min) was 0.84 ± 0.05 and 0.68 ± 0.20
mM, respectively. PTX added in B at 96 min
was 0.5 µg/ml arterial blood plasma. Mean fat pad weight was
5.2 ± 0.3 g (n = 9).