Download Effects of Fish Oil and Corn Oil Diets on

[CANCER RESEARCH 49, 1931-1936, April 15, 1989]
Effects of Fish Oil and Corn Oil Diets on Prostaglandin-dependent and
Myelopoiesis-associated Immune Suppressor Mechanisms of Mice
Bearing Metastatic Lewis Lung Carcinoma Tumors1
M. Rita I. Young2 and Melvin E. Young
Department if Research Services, Edward J. Hiñes,Jr., Veterans Administration Hospital, Hiñes,Illinois 60141 [M. K. 1. ¥.,M. E. Y.J, and Department of Pathology,
Loyola Univi rsity Stritch School of Medicine, Maywood, Illinois 601 S3 [M. R. I. Y.J
ABSTRACT
The effe -Is of a fish oil diet on the myelopoietic and immunological
parameters of normal mice and of mice bearing metastatic Lewis lung
carcinoma i LLC-C3) tumors were compared to the effects of a corn oil
or a mixec-fat rodent chow diet. This was studied soon after tumor
appearance, on Day 17, when immune suppression was mediated by
prostaglant in E2 (PGE2)-producing suppressor cells, and late in tumor
development, on Day 28 when immune suppression was associated with
myelopoiesis and the appearance of bone marrow-derived suppressor
cells whose activity was not dependent on PGE2. Feeding a fish oil diet
from Days 10 to 17 of tumor growth partially restored splenic T-cell
blastogene? is, reduced spleen cell secretion of PGE2, and alleviated
splenic suppressor activity. When fed from Days 21 to 28 of tumor
growth, a f sh oil diet neither restored T-cell blastogenesis nor alleviated
suppressor cell activity. The fish oil diet increased the frequency of
myeloid progenitor cells in normal mice and in mice bearing small or
large tumors. Concurrently, the fish oil diet stimulated the appearance of
bone marr jw-derived suppressor cells. When administered after the
establishm -nl of palpable primary tumors, a fish oil diet also increased
the formation of pulmonary lung nodules. In contrast to the fish oil
stimulatior of myelopoiesis and the associated suppressor cells, feeding
a corn oil diet to tumor-bearing mice during Days 21 to 28 after tumor
implantation reduced myelopoiesis and the presence of the associated
bone marrow suppressor cells. These data show that a fish oil diet can
minimize t le immune suppression in tumor bearers when suppression is
mediated by PGE2-producing suppressor cells, but can also induce mye
lopoietic siimulation leading to the appearance of bone marrow-derived
suppressor cells and increased tumor metastasis.
INTRODUCTION
Modul itors which regulate immune responses, in many in
stances, also influence myelopoiesis (1-9). Attention has been
focused on arachidonic acid metabolites, predominantly PGE2,3
as mediators of both immune suppression and as negative
regulators of myelopoiesis. PGE2 has been shown to be inhibi
tory to the activities of T-cells, B-cells, and macrophages (1014). In addition, PGE2 is myelosuppressive as it is inhibitory
to the g-owth of progenitor cells including monocytic and
granulocytic-monocytic CFC (1,2, 15).
Dietary fats may be capable of modifying both immunological
and mye opoietic parameters. Prostaglandin precursors, when
administered in the diet, P.O., or by s.c. injection, have resulted
in increased PGE2 production (16, 17). Dietary administration
of corn oil, which is rich in n-6 prostaglandin precursors, to
rats resulted in a reduced blastogenic response to PHA as
Receivec 9/28/88; revised 1/6/89; accepted 1/16/89.
The costs of publication of this article were defrayed in part by the payment
of page chi rges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This work was supported in part by the Medical Research Services of the
Veterans Administration and by Grant CA-45080 from the NIH.
2 To whom requests for reprints should be addressed, at Pathology Research
(151Z2), HiñesV.A. Hospital, Hiñes,IL 60141.
3The abbreviations used are: PGE2, prostaglandin E2; CFC, myeloid progeni
tor cells; PHA, phytohemagglutinin; EPA, eicosapentaenoic acid; CSF, colonystimulating factor; FBS, fetal bovine serum; Con A, concanavalin A; LLC-C3,
Lewis lung carcinoma, C-3 variant.
compared to that of rats fed a rodent chow diet (18). The
substitution of the dietary corn oil with increasing amounts of
fish oil, which is rich in n-3 polyunsaturated fats, increased
PHA responsiveness. Humans treated with EPA, a major n-3
polyunsaturated fat of fish oil, had increased T-cell responses
to PHA (19). EPA is a poor substrate for cyclooxygenase and
competitively inhibits the formation of dienoic prostaglandins
(20, 21). The administration of fish oil to mice reduced the
amount of PGE2 which their macrophages secreted (22). The
modulation of immune competence by corn oil or fish oil diets
has also been expressed in terms of their effects on growth of
transplanted tumors. Growth of mammary adenocarcinomas
was hastened in rodents which had been maintained on corn
oil diets and was reduced in animals fed fish oil diets (23, 24).
Studies of the effects of dietary polyunsaturated fats on tumor
growth have centered on the ability of the fats to modulate
PGE2 levels and, in turn, PGE2-dependent suppressor mecha
nisms. However, the suppressed immune competence in tumor
bearers has been associated with several different types of
immune suppressor mechanisms such as PGE2-secreting mac
rophages (25-27) and suppressor T-lymphocytes (28, 29). Re
cently, the suppressed immune competence of tumor bearers
has been associated with tumor stimulation of myelopoiesis
and the appearance of bone marrow-derived immune suppressor
cells (30-32). Tumors have been shown to stimulate myelo
poiesis by their secretion of CSFs (30, 32-36). Tumor bearers
have splenomegaly (30, 31, 37, 38), increased numbers of
myeloid CFC in the bone marrow and spleen (30, 31, 34, 39),
granulocytosis (34, 37), and reduced proportions of lympho
cytes (36, 40). The effects of dietary fats on myelopoiesis or on
the appearance of the associated bone marrow-derived suppres
sor cells have not been studied in either normal or tumorbearing animals.
Our previous characterizations of the immune suppressor
mechanisms in mice bearing metastatic LLC-C3 tumors have
shown that the tumor cells do not secrete immune suppressive
factors, such as PGE2 (41). Instead, the LLC-C3 tumors stim
ulate the sequential appearance of several immune suppressor
cell populations during the progressive growth of tumor. In
mice bearing small and medium sized LLC-C3 tumors, immune
suppression was associated with increased production of PGE2
by splenic adherent macrophages (27,42). In mice bearing large
LLC-C3 (>3 g), myelopoiesis was stimulated and, concurrently,
there was the presence of bone marrow-derived immune sup
pressor cells (30, 31). In the current study, the effects of diets
containing either corn oil, fish oil, or mixed fats on myelopoiesis
and on the two immune suppressor mechanisms of mice bearing
metastatic LLC-C3 tumors were investigated.
MATERIALS
AND METHODS
Mice. Six- to 8-wk-old male C57BL/6 mice were used for all studies.
The mice were obtained from Cumberland View Farms (Clinton, TN)
1931
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FATS, MYELOPOIESIS,
and then housed, in groups of 5 mice per cage, at the HiñesV. A.
animal research facility.
Medium. The medium used to culture tumor cells and for blastogenesis and suppressor cell assays was RPMI-1640 containing 100 units/
ml of penicillin, 100 ng/ml of streptomycin, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid buffer solution, 5 x IO"5 M 2-mercaptoethanol. 2 HIM L-glutamine, and 10% endotoxin-free FBS (Hyclone
Laboratories, Logan, UT).
Diets. Diets containing 5% fat were administered to tumor-bearing
or control mice between Days 10 and 17 or Days 21 and 28 of tumor
growth. The diets differed only in fat composition and not in fat
quantity. The following diets were used: fat-free basal diet mix contain
ing either 5% corn oil or 4% fish oil plus 1% corn oil; or rodent chow
containing 5% mixed fats. The basal diet was a vitamin- and mineralsupplemented fat-free mix (TD 84010, Teklad Test Diets, Madison,
WI). The fish oil (Zapata Haynie Corp., Reedville, VA), derived from
menhaden fish, was rich in eicosapentaenoic acid as well as in other n
3 polyunsaturated fatty acids. Corn oil, 1%, was added to this fish oil
diet as a source of essential fatty acids. Fresh food was given daily.
Characteristics of Tumor Cells (41). The cloned metastatic LLC-C3
cells were used in all studies. The LLC-C3 variant was chosen for this
study of host immune suppressor mechanisms of tumor bearers because
the tumor cells do not themselves secrete immune suppressive factors,
such as PGE2. Briefly, the LLC-C3 variant was isolated from a lung
nodule of a mouse bearing a s.c. implanted parental LLC tumor.
Injection of LLC-C3 cells s.c. typically results in formation of approx
imately 10 to IS metastatic lung nodules. Metastatic dissemination to
other sites has never been detected. In the currently described studies,
the LLC-C3 tumor cells were implanted into mice by dorsal s.c.
injection of 5 x 10' cells. Experimental diets were administered between
Days 10 and 17 after tumor injection, the first week after appearance
of a palpable tumor, or between Days 21 and 28. when tumors were
large. Assays were conducted on the last day of administration of
experimental diets.
Femoral Bone Marrow and Spleen Cellularity. Single cell suspensions
were prepared from the spleen and femoral bone marrow of each mouse
from at least 5 mice per group. The nucleated cells were counted on a
hemacytometer.
Soft Agar CFC Assay (9). Into each 35- x 10-mm tissue culture dish
were placed 7.5 x IO4bone marrow cells or 7.5 x IO5spleen cells in I
ml of semisolid supplemented RPMI-1640 medium containing 20%
FBS and 0.3% agar (Bacto Agar; Difco Laboratories, Detroit, MI).
Supernatant of pokeweed mitogen-stimulated spleen cells was used as
a CSF source at a concentration of 5% (43). This preparation of murine
CSF, which was free of known inhibitory molecules, stimulated colonies
containing mainly granulocytes and granulocytes plus macrophages.
The colonies (>50 cells) were counted after 7 days of culture.
T-I.ymphocyte Blastogenesis to Con A. Spleen cells were cultured for
3 days in microtiter plates at a density of 2 x IO5 cells in 0.2 ml of
medium containing 3 ^g/ml of Con A. For the last 18 h, l /iCi of [3H]thymidine was added. The amount of [3H]thymidine incorporated by
the spleen cells was measured in a liquid scintillation counter. All assays
were conducted in triplicate on individual mice from groups of at least
5 mice. Data were expressed as mean ±SEM (cpm) of 5 mice per
group assayed individually.
Suppressor Cell Assay. Suppressor cell activity was measured by the
capacity to suppress normal spleen cell blastogenesis to Con A. Sup
pressor cells were first irradiated with 2500 R. They were then added
to 2 x IO5normal responder spleen cells in a T-cell blastogenesis assay
AND SUPPRESSOR
CELLS
removed and frozen. A radioimmunoassay (New England Nuclear,
Boston, MA) was used to quantitate the PGE2 levels. Plasma was
diluted 1:20, and the supernatants were diluted 1:2 with assay buffer
prior to being added to the radioimmunoassay. Briefly, into each
polypropylene tube were mixed 0.1 ml of anti-PGE2, 0.1 ml of 12!IPGE2, and 0.1 ml of PGE2 standard or PGE2-containing sample. Tubes
were refrigerated overnight. A polyethylene glycol solution (16%, M,
6000) was added to precipitate immune complexes, and the radioactiv
ity in the precipitate was counted in a gamma counter. We have
previously shown that there is no nonspecific interference in this assay
since similar amounts of PGE2 were quantitated by this method in
diluted plasma and by radioimmunoassay of PGE2 which was purified
from plasma by high-performance liquid chromatography (44).
Tumor Growth and Metastasis. Mice which were implanted with
LLC-C3 tumor cells were maintained on a mixed-fat rodent chow diet
until the primary tumors became palpable at approximately 10 days.
These mice were then randomly divided into two groups of 15 mice
each. One group remained on a mixed-fat rodent chow diet, while the
other group was switched to a fish oil diet for the remaining 2.5 wk of
tumor growth. Diameters of primary tumors were measured during
tumor development. After 4 wk of tumor growth, all mice were sacri
ficed. The lungs were filled with a nigrosin solution, removed, and
washed in a mixture of 70% ethanol, 3.7% formaldehyde, and 5% acetic
acid. White metastatic nodules on the black lung background were
counted. Data were expressed as both the range and the median number
of lung nodules.
Analysis of Data. The significance of the differences between values
was determined by the Student / test. All myelopoietic and immunological data were expressed as the mean ±SD of triplicates or the mean
±SEM from at least 5 mice with triplicate determinations for each
mouse.
RESULTS
Effects of Fish Oil Diet, Corn Oil Diet, or Mixed-Fat Rodent
Chow on T-Lymphocyte Blastogenesis of Tumor-bearing Mice.
The effects of administering diets containing various fats on Tcell competence of normal mice or of mice bearing small or
large tumors were assessed (Fig. 1). The Con A blastogenic
response of normal mice was reduced after feeding a corn oil
diet (P < 0.001) and stimulated after feeding a fish oil diet (P
< 0.01) as compared to the response of mice fed a mixed-fat
rodent chow diet. In comparison to the response of control
mice, the Con A blastogenic response by spleen cells of mice
bearing LLC-C3 tumors and fed rodent chow was suppressed
at both 17 and 28 days after tumor implantation (P < 0.01).
On Day 17 after tumor implantation, the blastogenic response
200 -,
,
^
_
Chow
D
Corn
Fish
with 3 Mg/m' of Con A at a responder spleen cell:suppressor cell 1:1
ratio. In some experiments, indomethacin was also added to yield 5 x
10~6M(<0. l % ethanol). In all of the described experiments, the amount
of ['Hjthymidine which was incorporated in control cultures containing
only irradiated cells and Con A was less than 5000 cpm. Data were
expressed as mean ±SD (cpm) of triplicates.
Normals
LLC—C3
Normals
LLC—C3
Quantitation of PGE2 in Plasma or Secreted by Spleen Cells. Mice
Days 10-17
Days 21-28
were exsanguinated by retroorbital puncture, and the blood was placed
Fig.
Con A blastogenic response of spleen cells from normal mice and of
into IKp.u in coaled tubes. Plasma was collected and frozen until as
LLC-C3 tumor-bearing mice fed rodent chow, corn oil, or fish oil diets during
sayed for PGE2. Spleen cells were seeded into microtiter wells at a Days 10 to 17 or 21 to 28 of tumor growth. Spleen cell blastogenesis was assessed
density of 5 x 10V0.2 ml. After 4 h of incubation, supernatants were on the last day of diet administration. 1'ilK.thymidine.
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FATS, MYELOPOIESIS,
AND SUPPRESSOR
of tumor-tearing mice which had been fed a corn oil diet during
the precec ing 7 days was equally suppressed as that of mice
which had been fed mixed-fat chow. When, instead, a fish oil
diet was administered to the mice, the immune suppression was
abolished. The effect of administering the fish oil diet to mice
during I); ys 21 to 28 of tumor growth differed as it had no
immune-r>:storative effects on the T-lymphocyte responsiveness
to Con A. In fact, the blastogenic responses of tumor bearers
which were fed either rodent chow, corn oil diet, or fish oil diet
during Days 21 to 28 were equally suppressed.
Immune Suppressor Cell Activities of Normal or LLC-C3bearing M ice Fed Fish Oil or Rodent Chow Diets during Days
10 to 17 ifter Tumor Implantation. Studies were conducted to
support our previous investigations (27, 30, 42) of whether
spleen ani bone marrow cells of mice bearing small tumors
mediate their suppressive activities by a prostaglandin-dependent mechanism. At 17 days after tumor implantation, the spleen
cells of tu Tior-bearing mice fed rodent chow had more suppres
sor cell activity than did spleen cells of control normal mice (/'
< 0.001) (Table 1; Fig. 2). As shown in Table 1, this splenic
suppressor activity was prostaglandin dependent as it was
ablated by the addition of indomethacin. Tumor-induced bone
marrow sjppressor activity was not yet apparent on Day 17.
The effect of feeding a fish oil or rodent chow diet on the
suppressiwe activities of spleen and bone marrow cells was
assessed ¡
Fig. 2). The splenic suppressor activity of normal
mice fed a fish oil diet was slightly stimulated (P < 0.05) in
comparison to when a mixed-fat rodent chow diet was fed. The
administration of a fish oil diet to tumor bearers during Days
10 to 17 alleviated the splenic suppressor cell activity. In fact,
there was no difference in the suppressive activities of spleen
cells of n .trmal mice irr.vw.vtumor-bearing mice fed a fish oil
diet.
Since the immune suppressive activity of spleen cells obtained
from mie i at Day 17 after tumor implantation was indometh
acin sensitive, the amounts of PGE2 which were secreted by
spleen ce Is and which were in plasma of tumor-bearing mice
fed either rodent chow or fish oil diet were quantitated. Spleen
cells of tumor bearers fed a rodent chow diet secreted 53%
more (P < 0.01) PGE2 (490 ±23 pg of PGE2/106 cells/4 h)
than did spleen cells of control normal mice (323 ±17 pg of
PGE2/10i cells/4 h). The administration of a fish oil diet
reduced t tie amount of PGE2 which was secreted by spleen cells
of tumor bearers by 31% (P< 0.05) to 347 ±16 pg/106 cells/
4 h. Likewise, plasma PGE2 levels of tumor bearers were 2-fold
CELLS
higher (P < 0.001) than were plasma PGE2 levels of control
normal mice (Table 2). The administration of a fish oil diet
reduced the amounts of PGE2 in plasma of tumor bearers by
57% (P < 0.001) and of normal mice by 52% (P < 0.001).
During this same time period of Days 10 to 17 after tumor
implantation, the effects of a fish oil diet and a mixed-fat chow
diet on the immune regulatory activities of bone marrow cells
were compared. At Day 17, the tumor-induced bone marrow
suppressor cells had not yet become prominent in mice fed a
rodent chow diet (Table 1; Fig. 2). However, administration of
a fish oil diet to tumor bearers stimulated the appearance of
bone marrow suppressor cells.
Immune Suppressor Cell Activities of Normal or LLC-C3bearing Mice Fed Fish Oil or Rodent Chow Diets during Days
21 to 28 after Tumor Implantation. We previously showed that
the immune suppression in mice bearing large tumors was
associated with myelopoiesis and the consequential appearance
of bone marrow-derived suppressor cells first in the bone mar
row and then in the spleen (30, 31). At Day 28 after tumor
implantation, the bone marrow cells of tumor bearers exhibited
prominent suppressor activity (Table 1; Figs. 2 and 3). The
administration of a fish oil diet further exaggerated the degree
of bone marrow suppressor activity (P < 0.02) (Figs. 2 and 3).
In contrast, diet containing corn oil significantly reduced the
level of bone marrow suppressor activity (P < 0.05) (Fig. 3).
The suppressor activity of spleen cells from rodent chow-fed
mice bearing large tumors was also prominent (Table 1; Figs.
2 and 3). This immune suppressive activity was prostaglandin
independent as it was unaffected by the presence of indometh
acin (Table 1). Administration of either a fish oil or corn oil
diet between Days 21 and 28 of tumor growth did not alleviate
the tumor-induced splenic suppressor activity (Figs. 2 and 3).
In fact, the splenic suppressor activity of mice fed the mixedfat rodent chow diet or the fish or corn oil diets remained
comparable.
Modulation of Myeloid CFC Frequency and the Number of
Nucleated Cells in the Spleen and Femoral Bone Marrow of
Tumor Bearers Fed Diets Containing Various Fats. In mice
bearing large tumors, the appearance of bone marrow-derived
suppressor cells was associated with tumor stimulation of mye
lopoiesis; myelopoietic stimulation was not readily apparent in
mice bearing small tumors (30, 31). When a fish oil diet was
fed instead of rodent chow to mice during Days 10 to 17 after
tumor implantation, there resulted an increase in the frequency
of CFC per 7.5 x 10" bone marrow cells from 106 ±7 to 196
Table 1 Indomethacin sensitivity of tumor-induced suppressor cells
Normal spleen cells were cultured alone or with irradiated spleen or bone marrow cells obtained from normal mice or from mice bearing LLC-C3 tumors at Day
17 or 28 after tumor implantation. To the cultures were also added 3 ¿ig/mlof Con A and either diluent or 5 x 10"' M indomethacin.
Assay
day
cells17
marrow28
by normal spleen
andDiluent123,775cells with added cells
value"NSC
Added
None
Normal spleen
LLC-C3 spleen
Normal bonw marrow
LLC-C3 bone
±12,701*
120,778 ±1,209
64,645 ±8,461 (48)''
95,41 7 ±7,769 (23)
(30)136,793
86,976 ±8,535
23,835 ±10,406
127,785 ±17,499
118,400 ±12,586
122,316 ±2,732
±9,601140,1
116,286
NS
<0.01
<0.01
<0.02NSNS
18 ±4,274
None
±7,414
Normal spleen
138,362 ±7,056
141,623 ±1,722
LLC-C3 spleen
66,400 ±18,470(51)
69,321 ±19,817(49)
NS
<0.01NS
Normal bone marrow
101,665 ±5,565 (26)
141,855 ±11,188
LLC-C3 bone marrowBlastogenesis 49,850 ±9,270 (64)Indomethacin1 63,812 ±3,349 (53)P
" Signifirance of the difference between the blastogenic response of spleen cells in the presence versus absence of indomethacin.
* Mean :: SD of cpm of triplicates.
' NS, no i significant.
* Numlx rs in parentheses, significant (P < 0.05) suppressor cell activity shown as the percentage of suppression of normal spleen cell blastogenesis.
1933
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FATS, MYELOPOIESIS,
AND SUPPRESSOR
150
•¿
Chow
E3 Fish
Kl
°100
1
50
I
I
Kl
Norm LLC
Spleen
None
Days
Norm LLC
Bone morrow
Norm
LLC
Spleen
1O-17
Days
Added
Norm
LLC
Bone morrow
21-28
cells
Fig. 2. Suppressor cell activity of spleen cells or bone marrow cells of normal
or LLC-C3 tumor-bearing mice which were fed rodent chow or fish oil diet during
Days 10 to 17 or 21 to 28 of tumor growth. Suppressor cell activity was measured
by the capacity of cells to inhibit the Con A blastogenic response of normal spleen
cells. TdR, thymidine.
Table 2 Plasma PGE¡levels of tumor-bearing mice fed varying fat diets
Plasma l'( ,1 levels of normal or LLC-C3 tumor-bearing mice which were fed
rodent chow, corn oil, or fish oil diets during Days 10 to 17 or 21 to 28 of tumor
growth.
Plasma PGEj levels of mice
respectively, as compared to that for control normal mice (Fig.
4). The administration of a fish oil diet during Days 21 to 28
to normal and to tumor-bearing mice caused a respective in
crease in the frequency of splenic CFC by 63% (P < 0.01) and
36% (P < 0.02), and of bone marrow CFC by 34% (P < 0.02)
and 18% (P < 0.01). In contrast, administration of a corn oil
diet to normal and to tumor-bearing mice caused a respective
reduction in the frequency of splenic CFC by 40% (P < 0.02)
and 16% (not statistically significant), and of bone marrow
CFC by 15% (P < 0.02) and 18% (P < 0.02).
The effects of diets on the cellularity of the femoral bone
marrow and spleens of both normal mice and of tumor-bearing
mice were also measured (Fig. 5). Neither the tumor presence
nor the various diets caused significant alteration in the number
of nucleated cells in the femoral bone marrow. Hence, the fish
oil-induced increases or corn oil-induced decreases in femoral
CFC frequencies described above were similar in magnitude to
the changes in the absolute numbers of CFC per femur. In
contrast, the development of a large tumor in mice fed rodent
chow was associated with an increase in spleen cellularity by
2.1-fold (P< 0.01). The number of nucleated cells in the spleen
of tumor bearers was slightly reduced (not statistically signifi
cant) by the administration of a corn oil diet and was increased
by 77% (P < 0.05) following the administration of a fish oil
diet. Therefore, the splenic CFC-stimulatory effects of fish oil
bearers1099
day1728DietsRodent
chow
Fish
oilRodent
±67°
276
(52)*586
±62
CELLS
250
-,
200
-
±161
(57)535
626 ±74
chow
±58
±63
Corn oil
911 ±44(155)
1269± 118(237)
Fish oilNormals531347 ±79 (59)LLC-C3 367 ±96 (69)
" Mean ±SEM determined in triplicate for each mouse and shown as pg of
PGE2 per ml of plasma of 5 mice per group.
* Numbers in parentheses, significant (/' < 0.05) effects of the com oil or fish
oil diets on plasma 1'( 11 levels shown as the percentage of the value for mice fed
the mixed-fat rodent chow diet.
150 •¿
Chow
D Corn
E Fish
^H
100
o:
o
H
I
"i
^H
Y///Ì
Normals
LLC-C3
Normals
LLC-C3
^H
H
Spleen
cells
Bone
marrow
cells
Fig. 4. Frequency of myeloid progenitor cells (CFC) per 7.5 X IO4 bone
marrow cells or 7.5 x 1(1'spleen cells of normal or LLC-C3 tumor-bearing mice
which were fed rodent chow, corn oil, or fish oil diets during Days 21 to 28 of
tumor growth.
50 -
None
Normals
Spleen
LLC-C3
cells
Added
Normals
LLC-C3
Bone morrow
cells
cells
Fig. 3. Suppressor cell activity of spleen cells or bone marrow cells of normal
or LLC-C3 tumor-bearing mice which were fed rodent chow, corn oil, or fish oil
diets during Days 21 to 28 of tumor growth. Suppressor cell activity was measured
by the capacity of cells to inhibit the Con A blastogenic response of normal spleen
cells. TdR, thymidine.
±19 (P < 0.02). The effects of feeding rodent chow, fish oil, or
corn oil diets on the myelopoietic parameters of tumor bearers
were more extensively evaluated in mice bearing large tumors.
The frequency of CFC in the spleen and in the bone marrow of
tumor-bearing mice fed a rodent chow diet during Days 21 to
28 was increased 2.3-fold (P < 0.01) and 1.5-fold (P < 0.001),
«-»
Normals
Spleen
LLC-C3
cells
Normals
Bone
LLC-C3
marrow
cells
Fig. 5. Number of nucleated cells in the spleen (:<!()') or in the femoral bone
marrow (x IO") of normal or LLC-C3 tumor-bearing mice which were fed rodent
chow, com oil, or fish oil diets during Days 21 to 28 of tumor growth.
1934
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FATS, MYELOPOIESIS.
AND SUPPRESSOR
diets were magnified by the stimulatory effects on both the
splenic CFC frequency and the spleen cellularity.
Plasma 1 GK> Levels of Mice Bearing Large Tumors and Fed
Various Fai Diets. Plasma PGE2 levels of mice fed either corn
oil or fish cil diets during Days 21 to 28 of tumor growth were
measured and compared to the plasma PGE2 levels of mice fed
a mixed-fat rodent chow diet (Table 2). The concentrations of
PGE2 in the plasma of rodent chow-fed normal mice and of
mice bearir g large tumors were similar. The administration of
a corn oil diet from Days 21 to 28 elevated the plasma PGE2
levels of normal mice by 55% (P< 0.001) and of tumor-bearing
mice by 137% (P < 0.001). In contrast, the administration of
fish oil diet to either normal or tumor-bearing mice resulted in
a reductior in plasma PGE2 concentrations. The fish oil diet
reduced th>; plasma PGE2 level of normal mice by 41% (P <
0.01) and c f tumor-bearing mice by 31% (P< 0.02).
Effect of Fish Oil Diet on Growth of Subcutaneous and Melastatic Lung Tumors. The sizes of the primary s.c. tumors and
the number of lung nodules of mice which were placed on either
a fish oil d et or retained on a mixed-fat rodent chow diet were
measured. There were no differences in the sizes of the primary
tumors of mice which were fed the mixed-fat rodent chow versus
the fish oil diet from the time of tumor appearance. In contrast,
mice fed t le fish oil diet had approximately a 4-fold greater
number of lung nodules than did mice fed the rodent chow (P
< 0.001). The median number of lung nodules of mice fed the
rodent chow diet was 10 with a range of 6 to 20 nodules/mouse.
The median number of lung nodules of mice fed the fish oil
diet was 3Õ1
with a range of 34 to 58 nodules/mouse.
CELLS
probably due to a decrease in the amount of PGE2 produced by
splenic macrophages. In fact, at Day 17 after tumor implanta
tion, not only was the splenic suppressor activity abolished, but
the amount of PGE2 secreted by spleen cells of tumor bearers
fed a fish oil diet was also reduced.
In contrast to fish oil's abolishment of splenic suppressor
DISCUSSION
activity of mice bearing small tumors, the fish oil diet stimulated
the suppressive activity of bone marrow cells. The fish oil diet
also caused a stimulation of myelopoiesis in normal and tumorbearing mice. Since PGE2 is known to be a negative regulator
of myelopoiesis (1, 2, 15), this stimulation of bone marrow
suppressive activity in LLC-C3 tumor bearers was attributed to
a PGE2-reducing effect of fish oil in mice bearing this CSFsecreting tumor (30) allowing for additional stimulation of
myelopoiesis and the appearance of the associated bone mar
row-derived suppressor cells. This was indirectly supported by
previous studies showing that the reduction in PGE2 levels in
normal mice by indomethacin administration resulted in mye
lopoietic stimulation (47-49).
The effects of the corn oil diet in mice bearing large tumors,
when immune suppression was associated with myelopoiesis
and the appearance of bone marrow-derived suppressor cells,
were opposite of the effects of fish oil. Corn oil, being rich in
the «-6polyunsaturated fat precursors for prostaglandins, in
creased the levels of PGE2 and subsequently moderated mye
lopoiesis. The depression of myelopoiesis in mice bearing large
tumors was the probable explanation for the parallel diminish
ment in the presence of bone marrow-derived suppressor cells.
This is in agreement with our recent studies showing that
injection of a stable analogue of PGE2 into mice bearing large
LLC-C3 tumors reduced myelopoiesis and the associated sup
pressor cell activity (50). In contrast to corn oil's diminishment
Immune suppression in mice with progressively growing
LLC-C3 tumors is mediated by two sequentially appearing
immune sjppressor mechanisms. In mice bearing small or
medium sized tumors, immune suppression is mediated by
PGE2-secrîtingadherent macrophages (27, 42). When tumors
become la'ge, the macrophage production of PGE2 declines,
and the adherent macrophages are no longer immune suppressive. Instead, the development of large metastatic LLC-C3
tumors is accompanied by myelopoietic stimulation and the
appearanc; of bone marrow-derived suppressor cells, first in
the bone narrow and then in the spleen, resembling immature
cells of the monocyte lineage (30). In the present study, we
determine i how myelopoiesis, immune competence, and these
immune suppressor mechanisms were modulated in normal and
tumor-bearing mice by a corn oil diet, which is rich in n-6
polyunsattirated fat precursors, or a fish oil diet, which is rich
in the n-3 polyunsaturated EPA. The diets were all composed
of 5% fat )ut differed in the source of fat which was used.
The adriinistration of a corn oil diet to normal mice reduced
Con A blnstogenesis and stimulated the immune suppressive
activity of their spleen cells. This is in agreement with studies
by others showing that increased levels of dietary n-6 polyun
saturated ats resulted in increased splenic PGE2 production (6,
17). The immune suppressive activities of PGE2 are well known
(10-14, 45, 46). The corn oil diet had no effect on the T-cell
blastogenic response of mice bearing small tumors, when PGE2
secretion n spleen cells was already elevated and suppression
was medi ited by PGE2-producing macrophages. Substitution
of fish oil for the fat in the diet increased Con A blastogenesis
by spleen cells of normal mice and restored the blastogenic
response ay spleen cells of mice bearing small tumors. The
restoration in immune responsiveness by a fish oil diet was
of bone marrow suppressor activity of mice bearing large tu
mors, the corn oil diet had no effect on either splenic T-cell
blastogenesis or on splenic suppressor activity. This could have
been due to a shift in suppressor mechanisms from myelopoiesis-associated suppressor cells to PGE2-secreting suppres
sor cells. However, studies to evaluate this possibility have not
yet been conducted.
A variety of studies have been conducted to assess the effects
of corn oil or fish oil diets on the establishment and subsequent
growth of transplanted tumors in mice (17, 23, 24). Such studies
have generally shown that a corn oil diet increases the capacity
of a tumor to become established and then to grow, while a fish
oil diet decreases the capacity of a tumor to become established
and to grow. In contrast to these studies, we assessed the effects
of a fish oil versus a mixed-fat rodent chow diet on the growth
and metastasis of tumors which had already become established.
Switching mice to a fish oil diet from the time of appearance
of a palpable s.c. tumor had no effect on the size of the primary
tumor. However, the formation of lung nodules was increased
in mice which had been switched to a fish oil diet. The fish oil
stimulation of tumor metastasis is consistent with the fish oil
stimulation of myelopoiesis-associated immune suppressor cells
in tumor bearers.
In conclusion, the results of this study showed that, when fed
to normal mice or to tumor-bearing mice, fish oil diets were
myelopoiesis stimulatory, while corn oil diets were myelopoiesis
suppressive. In addition, this study showed the capacities of
these diets to modulate the two immune suppressor mecha
nisms which develop during progressive growth of LLC-C3
tumors in mice. The fish oil diet alleviated the splenic suppres
sor activity when it was mediated by the production of PGE2.
However, when immune suppression was associated with mye-
1935
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research.
FATS, MYELOPOIESIS,
AND SUPPRESSOR CELLS
lopoiesis-associated suppressor cells, a fish oil diet exacerbated
the extent of bone marrow suppressor activity, while a corn oil
diet alleviated the bone marrow suppressor cell activity.
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1936
Downloaded from cancerres.aacrjournals.org on August 9, 2017. © 1989 American Association for Cancer Research.
Effects of Fish Oil and Corn Oil Diets on
Prostaglandin-dependent and Myelopoiesis-associated Immune
Suppressor Mechanisms of Mice Bearing Metastatic Lewis Lung
Carcinoma Tumors
M. Rita I. Young and Melvin E. Young
Cancer Res 1989;49:1931-1936.
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