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Effects of indomethacin on moloney sarcoma virus-induced tumors
EFFECTS OF INDOMETHACIN MOLONEYSARCOMAVIRUS-INDUCED
ON TUMORS
John L. Humes, John J. Cupo, Jr. and Helen R. Strausser Department of Zoology and Physiology Rutgers University Newark, New Jersey 07102 ABSTRACT Chronic administration of indomethacln to weanling 20 day old) BALB/c mice inoculated with Moloney sarcoma virus MSV) delayed the onset of the tumor and suppressed the tumor growth. As expected, the prostaglandln (PG) levels associated with the MSV-inJected leg muscle of the indomethacin treated mice were greatly depressed when compared to the elevated PG content associated with the tumors of MSV injected control mice. There was no effect of indomethacln on the cyclic AMP or cyclic GMP levels. The elevated levels of PG and cyclic AMP associated with the tumor were found to parallel the tumor growth. Administration of antilymphocyte serum (ALS) to the mice at the time of MSV inoculation resulted in accelerated and enhanced tumor growth. Indomethacln treatment of these mice similarly suppressed the tumor growth, but less dramatically. ACKNOWLEDGMENTS The authors wish to thank Dr. F. A. Kuehl, Jr. and Dr. H. R. Behrman, Merck Institute for Therapeutic Research, for generous gifts of indomethacin, P G a n d PG antlsera. This study was supported by NIH Biomedical Sciences Support Grant No. RR7059. J. L. H. is on leave of absence from the Merck Institute for Therapeutic Research and supported by the Merck Sharp & Dohme Research Laboratories. Reprint reguests should be addressed to H. R. S.
PROSTAGLANDINS JUNE 25, 1974 VOL. 6 NO. 6
463
PROSTAGLANDINS INTRODUCTION Prostaglandlns, especially PGE2, have been shown to be elevated in Moloney sarcoma vlrus-induced tumors as compared with non-infected leg muscle (l~. Elevated levels of PGE2 have also been reported in sarcoma lSO tumors in mice (2). PG or PG-like activities have been demonstrated in such human neoplasms as carcinoma of the thyroid (3) and Kaposi's sarcoma (4). Elevated PGE plasma levels have been measured in patients with medullary carcinoma of the thyroid and neurablastoma (5,6). Increased amounts of FG have been reported in various neoplastic cell and organ culture studies. Elevated PGE 2 levels have been f o n d in BPS/P cells (2)~ in polyoma virus transformed baby-hamster-kidney fibroblasts ( 7 ) a n d in five clonal strains of HSDM 1 mouse tumor cells (8). Jaffe et al. has reported PGE s~nthesis in 3 tissue culture murine c e ~ n e s . In these cells, N~,02'-dibutyrylcyclic AMP elicited a marked increase in PGE production (9,10). This study was initiated to ascertain the effect of indomethacin on the induction and growth of the tumor as well as the effect of this FG inhibitor on the endogenous tumor concentration of PGs and cyclic nucleotides. As the induction and regression of vlrus-induced tumors may be a function of the host's immune systems, the effects of indomethacin were evaluated in anti-lymphocyte serum treated mice. MATERIALS AND METHODS Male B A ~ / c J mice were purchased from Jackson Laboratories, Bar Harbor, Maine at 19 days of age. One or two days later, the right hind leg was injected with a MSV preparation as descrlbed(1). The mice were maintained on Purine mouse chow and water ad lib. The diameters of both the MSV-inJected and control hind legs were determined with calipers to ascertain the pattern and amount of tumor growth. The measurements were performed every day, commencing 3-4 days after the MSV injection. Stock solutions of indomethacln, 5 n~/ml absolute ethyl alcohol, were diluted at the time of injection l:lO with a 15% gelatin solution. Beginning the day after the MSV inoculation, 0.i ml ( 5 0 ~ g m or ~ 5 m g ~ g ) was injected s.c. every other day throughout the experimental period. A control vehicle, a l:10 dilution of ethyl alcohol with 15% gelatin, was administered to control mice in an identical manner.
464
JUNE 25, 1974 VOL. 6 NO. 6
PROSTAGLANDINS
The isolation and partial purification of the PGs and cyclic nucleotldes were performed as described (1) and the quantltatlon of these substances were performed by radioimmunoassay (5,11,12). RESULTS The growth pattern of the MSV-Induced sarcoma in weanling mice is shown (Fig, 1A). The earliest manifestation of the tumor (a 2 m m l n c r e a s e in the diameter of the tumor bearing leg as compared to the control leg diameter) could be observed as early as 8 days after the virus inoculation. The tumor mass was maximum between days 10-15, after which time the tumors gradually regressed. The corresponding tumor growth pattern in ALS Immunosuppressed mice is shown in Fig. iB. Rapidly owing tumors appeared in the AIS treated mice at least hr. before small tumors were noted in the MSV-InJected control mice. The majority of the immunosuppressed mice died by day 14.
~
Indomethacln, administered every other day, significantly delayed the onset of the tumor (approx. 4 days) in both experimental protocols. In addition, this treatment produced a significant inhibition of the tumor growth (Fig. IA and 1B). The PG content of the MSV-lnduced sarcoma paralleled the growth of the tumor (Fig. 2). No tumors were observed on days 3 and 6; however, on day I0 the mean of the tumor leg diameters was greater than 3 times the mean of the diameters of the control legs. Control leg diameters remained at 4 mm throughout the experimental period. At this time, day i0, a significant elevation in the amount of PGE was noted as well as a small increase in the amount of PGF. The cyclic AMP levels associated with the sarcoma followed a s i m i l a r p a t t e r n as the increased amount of cyclic AMP was concomitantly found with the appearance of the tumor (Fig. 3). The elevated PGF content of the ~ V - I n d u c e d sarcoma was severely inhibited ~91%) by administration of Indomethacln and the corresponding PGE content was reduced 67%. There was no change in the levels of the cyclic nucleotldes (Table l).
JUNE 25, 1974
VOL. 6 NO. 6
465
PROSTAGLANDINS
A
B 15-
15 14 w o3 4t t,r I-~,,
i.d o3 -H o3 rr Lt.I I-uJ '.=:I
13 12 II -
~ i1 0 Z eli "'
II
~E
D I-14. 0 Z
76-
g
5
7 e
6
5 4
4
6
8
I0
12
I 14
1 16
t
18
....
t ......
I
~,,,
4
20
l 6
I .... I 8 I0
I 12
DAYS A F T E R M S V
DAYS A F T E R M S V
466
12
.UE9 nO 8-
8-
41
13
I0
I0
ne 0
14
Figure 1A.
The effect of Indomethacin on the growth of MSV-induced tumors in w e a n l i n g mice. Each group contained 12 mice.
Figure IB.
The effect of indomethacin on the growth Of MSV-induced tumors in ALS treated mice. Each group contained 9 mice. The diameters of the non-lnjected mice was 4 mm in all experimental groups. ~o o Control Vehicle ---x .... x - - Indomethacin
JUNE
25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS O3
~: O
5 -
D
O
4 -
t)
[---] PGF
n-
F"~ PGE
03 ~ Z Z 0 0 3 .-1
-
00-
//I V/I V/I ill vii v// fll V/I v// v/I V/I vii /11
g~2Z
o.
Z ...I t9
V/F V// V/A //A
I -
L//.d //A
I-O3 0
//A
0
Figure
2.
The
3
6 10 DAYS AFTER MSV
PGF and PGE content
of ~ S V - I n d u c e d
tumors.
Groups of 3 mice were s a c r i f i c e d at various times a n d the PG c o n t e n t of the ~ S V - i n J e c t e d leg (muscle or tumor) was determined.
JUNE 25, 1974
VOL. 6 NO. 6
467
PROSTAGLANDINS
50-
40 Z m
[ CONTROL • ] LEG ~:~ MOLONEY SARCOMA LEG
Io o. 30
E O.
=E cn 20 b.I _J
0 3~ Q. iii
I0 //,,
0
Figure B.
468
//t ///
4
111 ///
I
111
/// z// i// iz/ /// ///
I
I
I
6 9 DAYS AFTER MSV
13
The cycllc-AMP content of ~SV-Injected leg muscle and non-lnJected leg muscle. Groups of 4 mice were sacrificed at various times and the cycllc-AMP content determined.
JUNE 25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS
Table I
The Effect of Indomethacin on the Prostaglandin and Cyclic Nucleotide Content of Moloney Sarcoma Tumors
pMoleslmg Protein Moloney Sarcoma Tumors PGF
I. 15 + 0.33 (6)
Indomethacin-treated Moloney Sarcoma Tumors
Inhibition
p
0. 10 + 0.03 (6]
91%
<0.01 <0.01
PGE
13.8
+ 3.1
(61
3.3
± 0.6
(61
76"Io
Cyclic AMP
35.6
+- 6.4
~6)
34.2
+- 7.7
(6)
0
NS
0.43 ± 0.21 (5)
0
NS
Cyclic GMP
JUNE 25, 1974
0.63 -+- 0.33 (5)
VOL. 6 NO. 6
469
PROSTAGLANDINS
DISCUSSION MSV-induced murine sarcomas have been described as rhabdomyasarcomas and the growth of these tumors in BALB/c mice is well documented (13). The progress of the tumors varies with the age and the immune state of the animal. In the experiments reported here, the tumor bearing leg of the weanling mouse grows to approximately 3 times the diameter of the normal leg, and then regresses. In older mice (14), with a deficient immune response (15), the animal does not survive as the tumor grows and metastasizes to other organs. In the event that the immune system is suppressed, as with the use of X-irradiation (16), thymectomy (17), immunosuppressive steroids (18), anti-lymphocyte globulin (19), and as shown in the present experiments, the tumor grows progressively and undoubtedly causes the death of the animal. That part of the immune system mediating the regression of the tumor has been defined as beth humoral and cellular, probebly indicating the participation of both T and B cells. In fact, it has been shown that "MSV-induced tumors can be cured by lymphoid cells or serum From syngenelc or allogeneic donors specifically sensitized to MSV-induced tumor antigens" (20). The results reported here support the previous studies in which murine tumors have been shown to contain elevated amounts of PG ( l t 2 , 8 ) . The ability of PG, especially PGE, to stimulate cyclic AMP synthesis in a variety of normal cells and tissues (21), suggests that one of the roles of elevated PGE may be to elevate cyclic AMP levels in adjacent cells resulting in elevated cyclic AMP levels associated with the tumor. However, it should be noted that PG increases are associated with inflammatory conditions (22). The morphology of the MSV-induced tumor has previously been described (23) as a mixture of "inflammatory cells and granulation tissue that permeated subcutaneous tissues and muscle". Tumor regression was accompanied by a decrease in tumor cells and a progressive increase in the number of inflammatory cells (24). Histological studies of the MSV-induced tumor in the present experiments similarly demonstrated large masses of infiltrating leukocytes as well as large areas of inflammation. I ndomethacin is an established prostaglandin synthetase inhibitor (25,26,27) which results in anti-inflammatory action. As to be expected, indomethacin inhibited the elevated PG levels of the tumorous legs (91% inhibition of PGF and 76% inhibition of PGE). The inflammation of the tumor was considerably reduced as evidenced by histological examination and this reduction probably accounted for the fact that the tumor size was so considerably depressed. Similar results were obtained in the HSDM1 tumor, a tral~plantable mouse fibrosarcoma, following indomethacin treatment (8). On the other hand, Sykes and Maddox (2) found no significant reduction in sarcoma 180 weights in male Schneider mice following indomethacin treatment and concluded that it was possible to decrease PG levels without affecting tumor growth.
470
JUNE 25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS In another aspect of the present study, the effect of i ndomethacin immunosuppressed (ALS) tumor-bearing mice was investigated. The findings here support the theory that the immune system is necessary for tumor regression, but that inclomethacin can act e v e n in the absence of immune cells and antibodies to partially suppress tumor growth. Thus it would appear that PG acts to promote tumor growth and that the synergistic action of the immune system and indomethacln treatment suppresses tumor growth.
REFERENCES
1.
Humes, J. L. and Strausser, H . R . Prostaglandins and cyclic nucleotldes in Moloney sarcoma tumors. Prostaglandlns, ~: 183-196, 197~.
2.
Sykes, J. A. C. and Maddox, I. So Prostaglandln production by experimental tumors and effects of antl-lnflammatory compounds. Nature New Biol., 2ST: 59-61, 1972.
.
.
.
.
.
Williams, E. D., Karim, S. M. M. and Sandler, M. Prostaglandin secretion by medulla r~ carcinoma of the thyroid. The Lancet, ~: 22-25, 1968. Bhona, D., Hellier, K., Karim, S. M. M. and Pharm, B. Vasoactlve substances in Kaposi's sarcoma. Cancer, 2.~: 2BB-241, 1971. Jaffe, B. M., Behrman, H. R. and Parker, C. W. Radiolmmunoassay measurement of prostaglandln E, A and F in human plasma. J. Clln. Invest., 52: 398-405, 1973. Wolfe, L., Momer, O. and Rostworowskl, K. Prostaglandln levels in human body fluids. Clin. Res., 2._O0:925, 1972. HammarstrSm, S., Samuelsson, B. and BJursell, G. Prostaglandln levels in normal and transformed babyhamster-kidney flbroblasts. Nature New Biol., 2~3: 5o-51,
.
TashJlan, A. M., Jr,, Voelkel, E. F., Goldhaber, P. and Levlne, L. Prostaglandlns, calclummetabollsm and cancer. Fed.
.
1973.
Proc.,
33: 81-86,
1974.
Jaffe, B. M., Parker, C. W. and Philpott, G. W. Immunochemlcal measurements of prostaglandin or prostaglandin-llke activity from normal and neoplastic cultured tissues. Surg. Forum, 2_~2: 90-92, 1971.
JUNE 25, 1974 VOL 6 N O 6
471
PROSTAGLANDINS
I0.
Jaffe, B. M., Fhilpott, G. W., Hamprecht, B. and Parker, C . W . Prostaglandln production by cells in vitro. Advances in the Biosciences., ~: 179-181, 1973.
ii.
Behrman, H . R . Radlolmmunoassay of prostaglandins. The Physiologist, 14_: ll0-111, 1971.
12.
0rczyk, G. P. and Behrman, H . R . Ovulation blockage by aspirin or indomethacin -- in vitro evidence for a role of prostaglandin in gonadotrophln secretion. Prostaglandins,!: 3-20, 1972.
13.
Perk, K. and Moloney, J . B . Pathogenesis of a vlrus-lnduced rhabdomyosarcoma in mice. J. Nat. Cancer Inst., 37: 581-599, 1966.
14.
Strausser, H. ~. and Bober, L . A . Inhibition of tumor growth and survival of aged mice inoculated with Moloney tumor transplants and treated with endotoxin. Cancer Res., 32:2156-2159 , 1972.
15.
Strausser, H. R., Bober, L. A., Bucsl, R. A., Shillcock, J. A. and Goldstein, A . L . Stimulation of the hemagglutinln response of aged mice by cell free lymphoid extracts and bacterial endotoxins. Exptl. Gerontol., 6: 373-375, 1971.
16.
Fefer, A., McCoy, J. L. and Glynn, J . P . Induction and regression of primary Moloney sarcoma vlrus-induced tumors in mice. Cancer Res., 27: 1626-1631, 1967.
17.
Law, L . W . Studies of thymlc function with emphasis on the role of the thymus in oncogenesis. Cancer Res., 26: 551-574, 1966.
18.
Shachat, D. A., Fefer, A and Moloney, J . B . Effect of cortisone on oncogenesis by marine sarcoma virus (Moloney). Cancer Res., 28: 517-520, 1968.
19.
Vandeputte, M. Immunosuppresslon and cancer. Ann. Inst. Pasteur, !2~: 677-683, 1972.
20.
Fefer, A. Immunotherapy and chemotherapy of Moloney sarcoma vlrus-induced tumors in mice. Cancer Res., 29: 2177-2183, 1969.
21.
Kuehl, F. A., Jr., Cirillo, V. J., Ham, E. A. and Humes, J . L . The regulatory role of the prost~glandins on the cycllc-5',5'-AMP system. In S. Bergstrom (ed.), Advances in the Biosclences, Vol. 9, PP. 155-172. Oxford Pergamon Press, 1973.
472
JU-NE 25, ~974 VOL. 6 }WO. ~6
PROSTAGLANDINS 22.
Willis, A . L . Parallel assay of prostaglandin-like activity in rat inflammatory exudate by means of cascade superfusion. J. Pharm. Pharmac., 21.- 126-128, 1969.
23.
Stanton, M. F., Law, L. W. and Ting, R . C . Some biologic, immunogenic and morphologic effects in mice after infection with a murlne sarcoma virus. II. Morphologic Studies. J. Nat. Cancer Inst., 4_~0: 1113-1121, 1968.
24.
Fefer, A., McCoy, J. L., Perk, K. and Glynn, J. P. Immunologic, virologic and pathologic studies of regression of autochthonous Moloney sarcoma vlrus-induced tumors in mice. Cancer Res., 28: 1577-1585, 1968.
25.
Ferreira, S. H., Moncoda, S. and Vane, J. R. Indomethacin and aspirin abolish prostaglandln release from the spleen. Nature New Biol., 231: 237-239, 1971.
26.
Smith, J. B. and Willis, A . L . Aspirin selectively inhibits prostaglandln production in human platelets. Nature New Biol., 731: 235-237, 1971.
27.
Vane, J . R . Inhibition of prostaglandln synthetase as a mechanism of action for asplrln-llke drugs. Nature New Biol., 231: 232-235, 1971.
JUNE 25, 1974 VOL. 6 NO. 6
473
ON TUMORS
John L. Humes, John J. Cupo, Jr. and Helen R. Strausser Department of Zoology and Physiology Rutgers University Newark, New Jersey 07102 ABSTRACT Chronic administration of indomethacln to weanling 20 day old) BALB/c mice inoculated with Moloney sarcoma virus MSV) delayed the onset of the tumor and suppressed the tumor growth. As expected, the prostaglandln (PG) levels associated with the MSV-inJected leg muscle of the indomethacin treated mice were greatly depressed when compared to the elevated PG content associated with the tumors of MSV injected control mice. There was no effect of indomethacln on the cyclic AMP or cyclic GMP levels. The elevated levels of PG and cyclic AMP associated with the tumor were found to parallel the tumor growth. Administration of antilymphocyte serum (ALS) to the mice at the time of MSV inoculation resulted in accelerated and enhanced tumor growth. Indomethacln treatment of these mice similarly suppressed the tumor growth, but less dramatically. ACKNOWLEDGMENTS The authors wish to thank Dr. F. A. Kuehl, Jr. and Dr. H. R. Behrman, Merck Institute for Therapeutic Research, for generous gifts of indomethacin, P G a n d PG antlsera. This study was supported by NIH Biomedical Sciences Support Grant No. RR7059. J. L. H. is on leave of absence from the Merck Institute for Therapeutic Research and supported by the Merck Sharp & Dohme Research Laboratories. Reprint reguests should be addressed to H. R. S.
PROSTAGLANDINS JUNE 25, 1974 VOL. 6 NO. 6
463
PROSTAGLANDINS INTRODUCTION Prostaglandlns, especially PGE2, have been shown to be elevated in Moloney sarcoma vlrus-induced tumors as compared with non-infected leg muscle (l~. Elevated levels of PGE2 have also been reported in sarcoma lSO tumors in mice (2). PG or PG-like activities have been demonstrated in such human neoplasms as carcinoma of the thyroid (3) and Kaposi's sarcoma (4). Elevated PGE plasma levels have been measured in patients with medullary carcinoma of the thyroid and neurablastoma (5,6). Increased amounts of FG have been reported in various neoplastic cell and organ culture studies. Elevated PGE 2 levels have been f o n d in BPS/P cells (2)~ in polyoma virus transformed baby-hamster-kidney fibroblasts ( 7 ) a n d in five clonal strains of HSDM 1 mouse tumor cells (8). Jaffe et al. has reported PGE s~nthesis in 3 tissue culture murine c e ~ n e s . In these cells, N~,02'-dibutyrylcyclic AMP elicited a marked increase in PGE production (9,10). This study was initiated to ascertain the effect of indomethacin on the induction and growth of the tumor as well as the effect of this FG inhibitor on the endogenous tumor concentration of PGs and cyclic nucleotides. As the induction and regression of vlrus-induced tumors may be a function of the host's immune systems, the effects of indomethacin were evaluated in anti-lymphocyte serum treated mice. MATERIALS AND METHODS Male B A ~ / c J mice were purchased from Jackson Laboratories, Bar Harbor, Maine at 19 days of age. One or two days later, the right hind leg was injected with a MSV preparation as descrlbed(1). The mice were maintained on Purine mouse chow and water ad lib. The diameters of both the MSV-inJected and control hind legs were determined with calipers to ascertain the pattern and amount of tumor growth. The measurements were performed every day, commencing 3-4 days after the MSV injection. Stock solutions of indomethacln, 5 n~/ml absolute ethyl alcohol, were diluted at the time of injection l:lO with a 15% gelatin solution. Beginning the day after the MSV inoculation, 0.i ml ( 5 0 ~ g m or ~ 5 m g ~ g ) was injected s.c. every other day throughout the experimental period. A control vehicle, a l:10 dilution of ethyl alcohol with 15% gelatin, was administered to control mice in an identical manner.
464
JUNE 25, 1974 VOL. 6 NO. 6
PROSTAGLANDINS
The isolation and partial purification of the PGs and cyclic nucleotldes were performed as described (1) and the quantltatlon of these substances were performed by radioimmunoassay (5,11,12). RESULTS The growth pattern of the MSV-Induced sarcoma in weanling mice is shown (Fig, 1A). The earliest manifestation of the tumor (a 2 m m l n c r e a s e in the diameter of the tumor bearing leg as compared to the control leg diameter) could be observed as early as 8 days after the virus inoculation. The tumor mass was maximum between days 10-15, after which time the tumors gradually regressed. The corresponding tumor growth pattern in ALS Immunosuppressed mice is shown in Fig. iB. Rapidly owing tumors appeared in the AIS treated mice at least hr. before small tumors were noted in the MSV-InJected control mice. The majority of the immunosuppressed mice died by day 14.
~
Indomethacln, administered every other day, significantly delayed the onset of the tumor (approx. 4 days) in both experimental protocols. In addition, this treatment produced a significant inhibition of the tumor growth (Fig. IA and 1B). The PG content of the MSV-lnduced sarcoma paralleled the growth of the tumor (Fig. 2). No tumors were observed on days 3 and 6; however, on day I0 the mean of the tumor leg diameters was greater than 3 times the mean of the diameters of the control legs. Control leg diameters remained at 4 mm throughout the experimental period. At this time, day i0, a significant elevation in the amount of PGE was noted as well as a small increase in the amount of PGF. The cyclic AMP levels associated with the sarcoma followed a s i m i l a r p a t t e r n as the increased amount of cyclic AMP was concomitantly found with the appearance of the tumor (Fig. 3). The elevated PGF content of the ~ V - I n d u c e d sarcoma was severely inhibited ~91%) by administration of Indomethacln and the corresponding PGE content was reduced 67%. There was no change in the levels of the cyclic nucleotldes (Table l).
JUNE 25, 1974
VOL. 6 NO. 6
465
PROSTAGLANDINS
A
B 15-
15 14 w o3 4t t,r I-~,,
i.d o3 -H o3 rr Lt.I I-uJ '.=:I
13 12 II -
~ i1 0 Z eli "'
II
~E
D I-14. 0 Z
76-
g
5
7 e
6
5 4
4
6
8
I0
12
I 14
1 16
t
18
....
t ......
I
~,,,
4
20
l 6
I .... I 8 I0
I 12
DAYS A F T E R M S V
DAYS A F T E R M S V
466
12
.UE9 nO 8-
8-
41
13
I0
I0
ne 0
14
Figure 1A.
The effect of Indomethacin on the growth of MSV-induced tumors in w e a n l i n g mice. Each group contained 12 mice.
Figure IB.
The effect of indomethacin on the growth Of MSV-induced tumors in ALS treated mice. Each group contained 9 mice. The diameters of the non-lnjected mice was 4 mm in all experimental groups. ~o o Control Vehicle ---x .... x - - Indomethacin
JUNE
25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS O3
~: O
5 -
D
O
4 -
t)
[---] PGF
n-
F"~ PGE
03 ~ Z Z 0 0 3 .-1
-
00-
//I V/I V/I ill vii v// fll V/I v// v/I V/I vii /11
g~2Z
o.
Z ...I t9
V/F V// V/A //A
I -
L//.d //A
I-O3 0
//A
0
Figure
2.
The
3
6 10 DAYS AFTER MSV
PGF and PGE content
of ~ S V - I n d u c e d
tumors.
Groups of 3 mice were s a c r i f i c e d at various times a n d the PG c o n t e n t of the ~ S V - i n J e c t e d leg (muscle or tumor) was determined.
JUNE 25, 1974
VOL. 6 NO. 6
467
PROSTAGLANDINS
50-
40 Z m
[ CONTROL • ] LEG ~:~ MOLONEY SARCOMA LEG
Io o. 30
E O.
=E cn 20 b.I _J
0 3~ Q. iii
I0 //,,
0
Figure B.
468
//t ///
4
111 ///
I
111
/// z// i// iz/ /// ///
I
I
I
6 9 DAYS AFTER MSV
13
The cycllc-AMP content of ~SV-Injected leg muscle and non-lnJected leg muscle. Groups of 4 mice were sacrificed at various times and the cycllc-AMP content determined.
JUNE 25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS
Table I
The Effect of Indomethacin on the Prostaglandin and Cyclic Nucleotide Content of Moloney Sarcoma Tumors
pMoleslmg Protein Moloney Sarcoma Tumors PGF
I. 15 + 0.33 (6)
Indomethacin-treated Moloney Sarcoma Tumors
Inhibition
p
0. 10 + 0.03 (6]
91%
<0.01 <0.01
PGE
13.8
+ 3.1
(61
3.3
± 0.6
(61
76"Io
Cyclic AMP
35.6
+- 6.4
~6)
34.2
+- 7.7
(6)
0
NS
0.43 ± 0.21 (5)
0
NS
Cyclic GMP
JUNE 25, 1974
0.63 -+- 0.33 (5)
VOL. 6 NO. 6
469
PROSTAGLANDINS
DISCUSSION MSV-induced murine sarcomas have been described as rhabdomyasarcomas and the growth of these tumors in BALB/c mice is well documented (13). The progress of the tumors varies with the age and the immune state of the animal. In the experiments reported here, the tumor bearing leg of the weanling mouse grows to approximately 3 times the diameter of the normal leg, and then regresses. In older mice (14), with a deficient immune response (15), the animal does not survive as the tumor grows and metastasizes to other organs. In the event that the immune system is suppressed, as with the use of X-irradiation (16), thymectomy (17), immunosuppressive steroids (18), anti-lymphocyte globulin (19), and as shown in the present experiments, the tumor grows progressively and undoubtedly causes the death of the animal. That part of the immune system mediating the regression of the tumor has been defined as beth humoral and cellular, probebly indicating the participation of both T and B cells. In fact, it has been shown that "MSV-induced tumors can be cured by lymphoid cells or serum From syngenelc or allogeneic donors specifically sensitized to MSV-induced tumor antigens" (20). The results reported here support the previous studies in which murine tumors have been shown to contain elevated amounts of PG ( l t 2 , 8 ) . The ability of PG, especially PGE, to stimulate cyclic AMP synthesis in a variety of normal cells and tissues (21), suggests that one of the roles of elevated PGE may be to elevate cyclic AMP levels in adjacent cells resulting in elevated cyclic AMP levels associated with the tumor. However, it should be noted that PG increases are associated with inflammatory conditions (22). The morphology of the MSV-induced tumor has previously been described (23) as a mixture of "inflammatory cells and granulation tissue that permeated subcutaneous tissues and muscle". Tumor regression was accompanied by a decrease in tumor cells and a progressive increase in the number of inflammatory cells (24). Histological studies of the MSV-induced tumor in the present experiments similarly demonstrated large masses of infiltrating leukocytes as well as large areas of inflammation. I ndomethacin is an established prostaglandin synthetase inhibitor (25,26,27) which results in anti-inflammatory action. As to be expected, indomethacin inhibited the elevated PG levels of the tumorous legs (91% inhibition of PGF and 76% inhibition of PGE). The inflammation of the tumor was considerably reduced as evidenced by histological examination and this reduction probably accounted for the fact that the tumor size was so considerably depressed. Similar results were obtained in the HSDM1 tumor, a tral~plantable mouse fibrosarcoma, following indomethacin treatment (8). On the other hand, Sykes and Maddox (2) found no significant reduction in sarcoma 180 weights in male Schneider mice following indomethacin treatment and concluded that it was possible to decrease PG levels without affecting tumor growth.
470
JUNE 25, 1974
VOL. 6 NO. 6
PROSTAGLANDINS In another aspect of the present study, the effect of i ndomethacin immunosuppressed (ALS) tumor-bearing mice was investigated. The findings here support the theory that the immune system is necessary for tumor regression, but that inclomethacin can act e v e n in the absence of immune cells and antibodies to partially suppress tumor growth. Thus it would appear that PG acts to promote tumor growth and that the synergistic action of the immune system and indomethacln treatment suppresses tumor growth.
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