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Inhibition of growth and pulmonary metastasis of B16-F10 murine melanoma by N-1554, a polyprenyl phosphate
159
Cancer Letters, 57 (1991) 159-163 Elsevier Scientific Publishers Ireland
Ltd.
Inhibition of growth and pulmonary metastasis of Bl6-FlO murine melanoma by N-1554, a polyprenyl phosphate Y. Okamoto, Research
(Received
T. Ohwaki,
Center,
Nisshin
30 October
Hour
S. Kawase,
Milling
Co.
Ltd.,
Y. lshii, T. Hongyo, 5-3-l
Tsurugaoka,
Y. Tahara
Ohi-machi.
Saitama
354
and Y. Mishima (Japan)
1990)
received 8 February 1991) (Accepted 11 February 1991)
(Revision
Summary
Introduction
Antitumor effect of N-l 554 (cr-dihydrodecaprenyl phosphate containing eight trans internal isoprene residues) against B16-F10 melanoma in syngeneic C57BL/6 mice was examined. B16-F10 cells were inoculated into the footpad of mice and N-1554 was intraperitoneally administered after the inoculation. The drug significantly inhibited the tumor growth in the footpad and dramatically reduced the pulmonary metastasis from the tumor. The antitumor effect of N-1554 was atmost abolished when the immunosuppressant carrageenan or anti-asialo GM1 antibody was administered to mice. In addition, pretreatment of host mice with N-1554 reduced the growth subcutaneously inoculated B16-FlO of melanoma. These results suggest that enhancement of host immune system may be involved in the antitumor effect of N-1554.
Carbohydrate residues on cell-surface glycoconjugates have been shown to play important roles in tumor metastasis [3,6] and in interactions between host immune system and tumor cells [1,5,9,16]. In the previous reports, Okamoto et al. showed that a-dihydrodecaprenyl phosphate containing internal isoprene eight trans residues (N-1554) selectively stimulated Nglycosylation of proteins in cultured hepatoma cells 114,151 and enhanced the adhesiveness of the cells to the substratum [14] . Because reduced adhesiveness is a fundamental characteristic of malignant cells, it seemed possible that the changes in cell-surface properties by N-1554 may alter the in vivo behavior of tumor cells. In this study, we examined the effects of N-1554 on growth and spontaneous pulmonary metastasis of B16-FlO melanoma in syngeneic mice. Possible involvement of host immune system in the effect of N-1554 was also investigated.
Keywords:
N-1554;
B16-F10 melanoma; host immune system
tumor metastasis;
Materials Correspondence
to: Y.
Flour Milling Co. Ltd., gun, Saitama
354,
Okamoto,
5-3-l
Research Center, Tsurugaoka, Ohi-machi,
0 1991 Elsevier Scientific in Ireland
Publishers
Methods
Reagents N-1554 was prepared scribed [14]. RPMI 1640
Nisshin Iruma-
Japan.
0304-3835/91/$03.50 Published and Printed
and
Ireland
Ltd
as previously demedium and fetal
160
bovine serum were purchased from Gibco Grand Island, N.Y. CarLaboratories, rageenan was purchased from Sigma, St Louis, MO. Anti-asialo GM1 (aGM1) rabbit antiserum was obtained from Wako Pure Chemical, Osaka. Other chemicals were of analytical grade, obtained from commercial sources. B16-F10 cell B16-F10 mouse melanoma cells, a line selected in vivo for high pulmonary colonization [7], were kindly provided by Dr. H. Nakano (National Cancer Center, Tokyo). The cells were maintained in plastic dishes (diameter, 6 cm; Falcon No. 3002) in RPM1 1640 medium containing 10% (v/v) fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 pg/ml) Incubation was performed at 37°C in a humidified atmosphere of air/CO, (19: 1). The cells were passaged twice weekly by an exposure to 0.2% (w/v) trypsin in Ca*+, Mg’+-free phosphate-buffered saline (PBS). Mice Five-week-old male C57BL/6 mice were obtained from Shizuoka Agricultural Association for Laboratory Animals, Shizuoka and entered the experiments at the age of 6-7 weeks. They were kept under pathogen-free conditons and given food and water ad libitum. spontaneous pulmonary and metastasis of Bl6-F10 melanoma Mice were inoculated with 4 x lo5 B16-FlO cells in 0.05 ml of serum-free RPM1 1640 medium in the footpad. N-1554 suspended in about 0.2 ml of 1% (v/v) DMSO - 0.9% (w/v) NaCl was i.p. injected every other day from day 1 to 27 after the inoculation. Mice in the control group received the vehicle alone. The tumor-bearing legs were resected under ether anesthesia and the tumor weight was measured at day 28. At day 42, the mice were sacrificed and their lungs were excised and fixed in 10% (v/v) formaldehyde.
Metastatic nodules on lung surface were counted under a dissecting microscope. When the effect of the immunosuppressant, either carrageenan or anti-aGM1 antibody, was examined, B16-FlO cells were inoculated and N-1554 (10 mg/kg) was intraperitoneally injected to the mice as described above. And the mice were further treated with the immunosuppressant. Carrageenan was dissolved in 0.9% NaCl at 10 mg/ml by heating at 80-90°C and the solution was intraperitoneally injected to the mice (0.5 ml/mouse) at day 0, 10 and 20 after melanoma cell inoculation. Rabbit antiserum to aGM, was diluted 1:50 with PBS. The diluted antiserum was injected into tail vein of the mice (0.2 ml/mouse) at day 0 and later every 5 days up to day 25 after the inoculation. Subcutaneous growth of B16-FIO melanoma N-1554 suspended in about 0.2 ml of 1% (v/v) DMSO - 0.9% NaCI (w/v) was intraperitoneally injected to mice every other day for 7 times. Control mice received the vehicle alone. At day 1 after the final injection, the mice were subcutaneously inoculated with 5 x lo4 B16-FlO cells in 0.1 ml of serum-free RPM1 1640 medium in the right flank. The growth of the tumor at the inoculated site was estimated by measuring mean tumor diameter (mean of the longest and the shortest diameters). And survival times of the tumorbearing mice were recorded.
Growth
Statistical analysis Differences in the number of metastastic nodules were analyzed using Mann-Whitney U-test and, for all other assays, differences between experimental values were analyzed with Student’s t-test. Results
Antitumor effect of N-1554 against B16-FlO melanoma was examined. In Table I, the effect of i.p. administration of N-1554 on the growth and pulmonary metastasis of the tumor is
161
either of the immunosuppressants caused little effect on the tumor weight in control mice. In N-1554-treated mice, both carrageenan and anti-aGMi antibody increased the tumor weight almost to control levels. The metastastic nodules on lung surface in control mice were significantly increased by anti-aGMi antibody but not by carrageenan. The metastatic nodules in N-1554-treated mice were also significantly incresed by anti-aGMi antibody and less significantly by carrageenan. As a result, the differences in the number of metastatic nodules between control and N-1554-treated mice became smaller by the administration of the immunosuppressants. Effect of pretreatment of host mice with N-1554 on the growth of S.C. inoculated B16-FlO melanoma was examined. When mice were administered with N-1554 before the inoculation of B16-FlO cells, the growth of the tumor was visibly reduced (for example, the mean tumor diameters at 16 days after the inoculation were 11.3 f 0.9 cm in the control mice and 6.5 f 2.0 cm in the mice treated with 10 mg/kg N-1554). And the mean survival time of the tumor-bearing mice was significantly prolonged by the pretreatment with N-1554 as shown in Table III.
shown. N-1554 significantly inhibited the growth of the tumor in the foot pad at the doses 2.5-20 mg/kg. The greatest inhibition of the growth (93%) was observed at 5 mg/kg. Moreover, the drug dramatically reduced the pulmonary metastasis from the tumor. Number of the mice with metastasis was dose-dependently decreased at 2.5-20 mg/kg and no metastasis was formed at 20 mg/kg. Also number of metastatic nodules formed on lung surface was significantly decreased. Similar results were obtained by intravenous administration of N-1554 in an oily emulsion (data not shown). Because N-1554 has only a little inhibitory effect on the proliferation of B16-FlO cells in vitro, the inhibition of the tumor growth in vivo by the drug was suggested to be caused by some changes in interactions between host immune system and the tumor. Accordingly, effects of immunosuppressants on N- 1554induced inhibition of growth and pulmonary metastasis of B16-FlO tumor were examined. Carrageenan and anti-aGMi antibody were used as inhibitors of macrophage function (2,121 and natural killer cell function [ 10,111, respectively. As shown in Table II, administration of
Table 1.Effect of intraperitoneal N- 1554 (mgbg)
0 2.5 5 10 20
administration
Pulmonary
Tumor weight(g)
1.13 zt 0.16” 0.37 f O.Hd 0.08 f 0.03d 0.20 zrz O.lOd 0.40 f 0.19’
of N-1554
(lOO)b (33) (7) (18) (35)
“Values are the means + S.E.M. bValues in parentheses are the percentages ‘P < 0.05, compared with the control. dP < 0.01, compared with the control.
on growth and pulmonary
metastasis
metastasis
No. of mice with metastasis
No. of metastatic nodules/mouse
14/18 5/10 3/15 l/15 o/10
13.1 5.2 0.8 0.9
of the controls.
zt f * f 0
8.6” (lOO)b 3.8 (40) 0.6d (6) 0.9d (7) (0)
of B16-FlO
tumor.
162 Table
II. Effects of immunosuppressants
on N-1554induced
inhibition of growth and pulmonary
metastasis
of B16-FlO
tumor. Treatment
Pulmonary
Tumor weight(g)
metastasis
No. of mice with metastasis Control Control
f
0.12”
0.83
* 0.07
0.93 0.21
f 0.15 zt 0.05d (18)b
0.76
ztz 0.12’
0.95
f
9/10
6.2 zt 4.1”
6/10
4.0 f
+ CA
Control + aGM, N- 1554 N-1554 + CA N-1554 aGM,
1.17
No. of metastatic nodules/mouse
lO/lO 2/10
24.1 + 6.9’ 0.2 f O.ld (3jb
5/10
(92)
0.2
3.0 f
1.3 (75)
+ 0.11’ (102)
9/10
14.5 + 5.1’ (60)
CA and aGM, in the Table indicate carrageenan and anti-asialo GM, antibody, respectively. “Values are the means + S.E.M. bValues in parentheses are the percentages of the control or of the immunosuppressant-treated ‘P < 0.05, compared with the control. dP < 0.01, compared with the control. ‘P < 0.05, compared with the mice treated only with N-1554. ‘P < 0.01, compared with the mice treated only with N-1554.
Discussion
N-1554
was not dose-dependent, that is, the doses of N-1554 higher than 10 mg/kg somewhat weakened the inhibitory effect of the drug. It is conceivable that some adverse effect of N-1554 on the host immune system may weaken the growth inhibitory effect of the drug because a toxicological study showed that growth
In the present study, we found that i.p. adof 2.5-20 mg/kg N-1554 ministration markedly inhibits the growth and pulmonary metastasis of B16-FlO melanoma in syngeneic mice. The inhibitory effect on the tumor
Table
111. Effect of pretreatment
with N-1554
on survival time of B16-FlO-bearing
Median
Mean
32.5 39.0 43.0
31.9 40.6 47.2
mice 70-day survivor
Survival time (days)
(mg/kg)
0 5 10
control.
+ S.E.M. zt 2.5 f 3.1b * 4.7’
T/C(%)” 100 127 148
0 0 1
For each experimental group, 10 mice were used. aThese values are calculated based on mean survival times of the control and the N-1554.treated bP < 0.05, compared with the control. ‘P < 0.01, compared with the control.
mice.
163
daily administration of 20 mg/kg N-1554 to normal mice caused the decrease in the ratio of lymphocytes in white blood cells (data not shown). Differing from the tumor growth, the incidence of the tumor metastasis to the lung was dose-dependently decreased by the administration of N-1554 at 2.5-20 mg/kg. These results suggest that the inhibition of tumor metastasis by N-1554 is not merely due to a secondary effect following the inhibition of tumor growth but also due to a primary effect of the drug. Some compounds capable of modifying oligosaccharide structures have been shown to inhibit experimental pulmonary metastasis of tumor cells [4,8,13]. It seems possible that changes in tumor cell surface properties via increased N-glycosylation may be related to the antimetastatic activity of N-1554. Administration of the immunosuppressants carrageenan or anti-aGM, antibody to the host mice abolished the growth inhibitory effect of N-1554 against B16-FlO melanoma. It was suggested that intact function of macrophages and natural killer cells is the prerequisite for the antitumor action of N-1554. In addition, pretreatment of host mice with N-1554 reduced the growth of subcutaneously inoculated B16-FlO melanoma and prolonged the survival time of the tumor-bearing mice. The data suggested that the enhancement of antitumor potential of host immune system may be involved in the effect of N-1554. For further clarifying the mechanism of the antitumor action of N-1554, it may be important to examine the direct effect of N-1554 on the host tumor surveillance system. In conclusion, the present study showed the noteworthy antitumor effect of N-1554 against B16-FlO melanoma. And it was suggested that the effect may be largely mediated by the enhancement of host immune system. References 1
Ahrens. P.B. and Ankel. H. (1987) The role of asparagine-linked carbohydrate in natural killer cellmediated cytolysis. J. Biol. Chem., 262, 7575-7579.
Catanzaro, P.J., Schwarz, H.J. and Graham, R.C. (1971) Spectrum and possible mechanism of carraqgeenan cytotoxicity. Am. J. Pathol.. 64, 387-404. Collard, J.G., Bikker, A., Rivier, G.L., Bolsher, J.G.M. and Roose, E. (1986) Cell surface sialic acid and the metastatic potential of T-cell hybridomas. Cancer Res., 46, 3521-3527. J.W. (1986) Effect of swainsonine and Dennis, polyinosinic:polycytidylic acid on murine tumor cell growth and metastasis. Cancer Res.. 46. 5131-5136. Dennis, J.W. and Laferte, S. (1985) Recognition of
7 8
9
10
11
12
13
14
15
16
asparagine-linked oligosaccharides on murine tumor cells by natural killer cells. Cancer Res., 45, 6034-6040. Dennis, J. W., Laferte, S., Waghorne. C., Breitman, M.L. and Kerbel. R.S. (1987) fi 1-6 Branching of Asn-linked oligosaccharides is directly associated with metastasis. Science, 236, 582-585. Fidler, I.J. (1973) Selection of successive tumor lines for metastasis. Nat. New Biol., 24, 148-149. Humphries, M.J., Matsumoto. K., White, S.L. and Olden, K. (1986) Oiigosaccharide modification by swainsonine treatment inhibits pulmonary colonization by B16-FIO murine melanoma cells. Proc. Natl. Acad. Sci. U.S.A., 83, 1752- 1756. Joshi, S.S., Tilden, P.A.. Jackson, J.D., Sharp, J.G. and Brunson. K.W. (1987) Cell surface properties associated with malignancy of metastatic large cell lymphoma cells. Cancer Res., 47, 3551-3557. Kalland. T. (1986) Effects of immunomodulator on growth and metastasis of the murine melanoma. Cancer Res.. 46. 3018-3022.
LS 2616 B16-F10
Kasai, M.. Yoneda. T., Habu, S., Maruyama. Y.. Okumura, K. and Tokunaga, T. (1981) In viva effect of anti-asialo GM antibody on natural killer cell activity. Nature (Land.), 291, 334-337. Keller, R. (1976) Promotion of tumor growth in viva by anJ. Natl. Cancer Inst., 57, timacrophage agents. 1355-1361. Kijima-Suds, I., Miyamoto, Y., Toyoshima, S.. ftoh. M. and Osawa, T. (1986) Inhibition of experimental pulmonary metastasis of mouse colon adenocarcinoma 26 sublines by a sialic acid:nucleoside conjugate having sialyltransferase inhibiting activity. Cancer Res., 46. 858-862. Okamoto. Y.. Murakami, K., Tahara, Y., Fukawa, H. and Akamatsu. N. (1985) Stimulation of protein glycosylation in cultured hepatoma ceils by polyprenyl phosphates. Jpn. J. Cancer Res., 76, 760-770. Okamoto, Y.. Murakami. K., Tahara, Y., Fukawa. H. and Akamatsu, N. (1986) o-Dihydrodecaprenyl phosphate as a sugar carrier in the membrane of AH 7OBtc hepatoma cells. Int. J. Biochem.. 18. 175-178. Yoshida. M., Gallik, G.E.. Irimura, T. and Nicolson, G.L. (1987) Modification of cell surface glycoprotein, macrophage cytostasis. and blood-borne metastatic properties of the murine RAW117 large cell lymphoma by virus superinfection. Cancer Res.. 47. 2558-2562.
Cancer Letters, 57 (1991) 159-163 Elsevier Scientific Publishers Ireland
Ltd.
Inhibition of growth and pulmonary metastasis of Bl6-FlO murine melanoma by N-1554, a polyprenyl phosphate Y. Okamoto, Research
(Received
T. Ohwaki,
Center,
Nisshin
30 October
Hour
S. Kawase,
Milling
Co.
Ltd.,
Y. lshii, T. Hongyo, 5-3-l
Tsurugaoka,
Y. Tahara
Ohi-machi.
Saitama
354
and Y. Mishima (Japan)
1990)
received 8 February 1991) (Accepted 11 February 1991)
(Revision
Summary
Introduction
Antitumor effect of N-l 554 (cr-dihydrodecaprenyl phosphate containing eight trans internal isoprene residues) against B16-F10 melanoma in syngeneic C57BL/6 mice was examined. B16-F10 cells were inoculated into the footpad of mice and N-1554 was intraperitoneally administered after the inoculation. The drug significantly inhibited the tumor growth in the footpad and dramatically reduced the pulmonary metastasis from the tumor. The antitumor effect of N-1554 was atmost abolished when the immunosuppressant carrageenan or anti-asialo GM1 antibody was administered to mice. In addition, pretreatment of host mice with N-1554 reduced the growth subcutaneously inoculated B16-FlO of melanoma. These results suggest that enhancement of host immune system may be involved in the antitumor effect of N-1554.
Carbohydrate residues on cell-surface glycoconjugates have been shown to play important roles in tumor metastasis [3,6] and in interactions between host immune system and tumor cells [1,5,9,16]. In the previous reports, Okamoto et al. showed that a-dihydrodecaprenyl phosphate containing internal isoprene eight trans residues (N-1554) selectively stimulated Nglycosylation of proteins in cultured hepatoma cells 114,151 and enhanced the adhesiveness of the cells to the substratum [14] . Because reduced adhesiveness is a fundamental characteristic of malignant cells, it seemed possible that the changes in cell-surface properties by N-1554 may alter the in vivo behavior of tumor cells. In this study, we examined the effects of N-1554 on growth and spontaneous pulmonary metastasis of B16-FlO melanoma in syngeneic mice. Possible involvement of host immune system in the effect of N-1554 was also investigated.
Keywords:
N-1554;
B16-F10 melanoma; host immune system
tumor metastasis;
Materials Correspondence
to: Y.
Flour Milling Co. Ltd., gun, Saitama
354,
Okamoto,
5-3-l
Research Center, Tsurugaoka, Ohi-machi,
0 1991 Elsevier Scientific in Ireland
Publishers
Methods
Reagents N-1554 was prepared scribed [14]. RPMI 1640
Nisshin Iruma-
Japan.
0304-3835/91/$03.50 Published and Printed
and
Ireland
Ltd
as previously demedium and fetal
160
bovine serum were purchased from Gibco Grand Island, N.Y. CarLaboratories, rageenan was purchased from Sigma, St Louis, MO. Anti-asialo GM1 (aGM1) rabbit antiserum was obtained from Wako Pure Chemical, Osaka. Other chemicals were of analytical grade, obtained from commercial sources. B16-F10 cell B16-F10 mouse melanoma cells, a line selected in vivo for high pulmonary colonization [7], were kindly provided by Dr. H. Nakano (National Cancer Center, Tokyo). The cells were maintained in plastic dishes (diameter, 6 cm; Falcon No. 3002) in RPM1 1640 medium containing 10% (v/v) fetal bovine serum, penicillin (100 units/ml) and streptomycin (100 pg/ml) Incubation was performed at 37°C in a humidified atmosphere of air/CO, (19: 1). The cells were passaged twice weekly by an exposure to 0.2% (w/v) trypsin in Ca*+, Mg’+-free phosphate-buffered saline (PBS). Mice Five-week-old male C57BL/6 mice were obtained from Shizuoka Agricultural Association for Laboratory Animals, Shizuoka and entered the experiments at the age of 6-7 weeks. They were kept under pathogen-free conditons and given food and water ad libitum. spontaneous pulmonary and metastasis of Bl6-F10 melanoma Mice were inoculated with 4 x lo5 B16-FlO cells in 0.05 ml of serum-free RPM1 1640 medium in the footpad. N-1554 suspended in about 0.2 ml of 1% (v/v) DMSO - 0.9% (w/v) NaCl was i.p. injected every other day from day 1 to 27 after the inoculation. Mice in the control group received the vehicle alone. The tumor-bearing legs were resected under ether anesthesia and the tumor weight was measured at day 28. At day 42, the mice were sacrificed and their lungs were excised and fixed in 10% (v/v) formaldehyde.
Metastatic nodules on lung surface were counted under a dissecting microscope. When the effect of the immunosuppressant, either carrageenan or anti-aGM1 antibody, was examined, B16-FlO cells were inoculated and N-1554 (10 mg/kg) was intraperitoneally injected to the mice as described above. And the mice were further treated with the immunosuppressant. Carrageenan was dissolved in 0.9% NaCl at 10 mg/ml by heating at 80-90°C and the solution was intraperitoneally injected to the mice (0.5 ml/mouse) at day 0, 10 and 20 after melanoma cell inoculation. Rabbit antiserum to aGM, was diluted 1:50 with PBS. The diluted antiserum was injected into tail vein of the mice (0.2 ml/mouse) at day 0 and later every 5 days up to day 25 after the inoculation. Subcutaneous growth of B16-FIO melanoma N-1554 suspended in about 0.2 ml of 1% (v/v) DMSO - 0.9% NaCI (w/v) was intraperitoneally injected to mice every other day for 7 times. Control mice received the vehicle alone. At day 1 after the final injection, the mice were subcutaneously inoculated with 5 x lo4 B16-FlO cells in 0.1 ml of serum-free RPM1 1640 medium in the right flank. The growth of the tumor at the inoculated site was estimated by measuring mean tumor diameter (mean of the longest and the shortest diameters). And survival times of the tumorbearing mice were recorded.
Growth
Statistical analysis Differences in the number of metastastic nodules were analyzed using Mann-Whitney U-test and, for all other assays, differences between experimental values were analyzed with Student’s t-test. Results
Antitumor effect of N-1554 against B16-FlO melanoma was examined. In Table I, the effect of i.p. administration of N-1554 on the growth and pulmonary metastasis of the tumor is
161
either of the immunosuppressants caused little effect on the tumor weight in control mice. In N-1554-treated mice, both carrageenan and anti-aGMi antibody increased the tumor weight almost to control levels. The metastastic nodules on lung surface in control mice were significantly increased by anti-aGMi antibody but not by carrageenan. The metastatic nodules in N-1554-treated mice were also significantly incresed by anti-aGMi antibody and less significantly by carrageenan. As a result, the differences in the number of metastatic nodules between control and N-1554-treated mice became smaller by the administration of the immunosuppressants. Effect of pretreatment of host mice with N-1554 on the growth of S.C. inoculated B16-FlO melanoma was examined. When mice were administered with N-1554 before the inoculation of B16-FlO cells, the growth of the tumor was visibly reduced (for example, the mean tumor diameters at 16 days after the inoculation were 11.3 f 0.9 cm in the control mice and 6.5 f 2.0 cm in the mice treated with 10 mg/kg N-1554). And the mean survival time of the tumor-bearing mice was significantly prolonged by the pretreatment with N-1554 as shown in Table III.
shown. N-1554 significantly inhibited the growth of the tumor in the foot pad at the doses 2.5-20 mg/kg. The greatest inhibition of the growth (93%) was observed at 5 mg/kg. Moreover, the drug dramatically reduced the pulmonary metastasis from the tumor. Number of the mice with metastasis was dose-dependently decreased at 2.5-20 mg/kg and no metastasis was formed at 20 mg/kg. Also number of metastatic nodules formed on lung surface was significantly decreased. Similar results were obtained by intravenous administration of N-1554 in an oily emulsion (data not shown). Because N-1554 has only a little inhibitory effect on the proliferation of B16-FlO cells in vitro, the inhibition of the tumor growth in vivo by the drug was suggested to be caused by some changes in interactions between host immune system and the tumor. Accordingly, effects of immunosuppressants on N- 1554induced inhibition of growth and pulmonary metastasis of B16-FlO tumor were examined. Carrageenan and anti-aGMi antibody were used as inhibitors of macrophage function (2,121 and natural killer cell function [ 10,111, respectively. As shown in Table II, administration of
Table 1.Effect of intraperitoneal N- 1554 (mgbg)
0 2.5 5 10 20
administration
Pulmonary
Tumor weight(g)
1.13 zt 0.16” 0.37 f O.Hd 0.08 f 0.03d 0.20 zrz O.lOd 0.40 f 0.19’
of N-1554
(lOO)b (33) (7) (18) (35)
“Values are the means + S.E.M. bValues in parentheses are the percentages ‘P < 0.05, compared with the control. dP < 0.01, compared with the control.
on growth and pulmonary
metastasis
metastasis
No. of mice with metastasis
No. of metastatic nodules/mouse
14/18 5/10 3/15 l/15 o/10
13.1 5.2 0.8 0.9
of the controls.
zt f * f 0
8.6” (lOO)b 3.8 (40) 0.6d (6) 0.9d (7) (0)
of B16-FlO
tumor.
162 Table
II. Effects of immunosuppressants
on N-1554induced
inhibition of growth and pulmonary
metastasis
of B16-FlO
tumor. Treatment
Pulmonary
Tumor weight(g)
metastasis
No. of mice with metastasis Control Control
f
0.12”
0.83
* 0.07
0.93 0.21
f 0.15 zt 0.05d (18)b
0.76
ztz 0.12’
0.95
f
9/10
6.2 zt 4.1”
6/10
4.0 f
+ CA
Control + aGM, N- 1554 N-1554 + CA N-1554 aGM,
1.17
No. of metastatic nodules/mouse
lO/lO 2/10
24.1 + 6.9’ 0.2 f O.ld (3jb
5/10
(92)
0.2
3.0 f
1.3 (75)
+ 0.11’ (102)
9/10
14.5 + 5.1’ (60)
CA and aGM, in the Table indicate carrageenan and anti-asialo GM, antibody, respectively. “Values are the means + S.E.M. bValues in parentheses are the percentages of the control or of the immunosuppressant-treated ‘P < 0.05, compared with the control. dP < 0.01, compared with the control. ‘P < 0.05, compared with the mice treated only with N-1554. ‘P < 0.01, compared with the mice treated only with N-1554.
Discussion
N-1554
was not dose-dependent, that is, the doses of N-1554 higher than 10 mg/kg somewhat weakened the inhibitory effect of the drug. It is conceivable that some adverse effect of N-1554 on the host immune system may weaken the growth inhibitory effect of the drug because a toxicological study showed that growth
In the present study, we found that i.p. adof 2.5-20 mg/kg N-1554 ministration markedly inhibits the growth and pulmonary metastasis of B16-FlO melanoma in syngeneic mice. The inhibitory effect on the tumor
Table
111. Effect of pretreatment
with N-1554
on survival time of B16-FlO-bearing
Median
Mean
32.5 39.0 43.0
31.9 40.6 47.2
mice 70-day survivor
Survival time (days)
(mg/kg)
0 5 10
control.
+ S.E.M. zt 2.5 f 3.1b * 4.7’
T/C(%)” 100 127 148
0 0 1
For each experimental group, 10 mice were used. aThese values are calculated based on mean survival times of the control and the N-1554.treated bP < 0.05, compared with the control. ‘P < 0.01, compared with the control.
mice.
163
daily administration of 20 mg/kg N-1554 to normal mice caused the decrease in the ratio of lymphocytes in white blood cells (data not shown). Differing from the tumor growth, the incidence of the tumor metastasis to the lung was dose-dependently decreased by the administration of N-1554 at 2.5-20 mg/kg. These results suggest that the inhibition of tumor metastasis by N-1554 is not merely due to a secondary effect following the inhibition of tumor growth but also due to a primary effect of the drug. Some compounds capable of modifying oligosaccharide structures have been shown to inhibit experimental pulmonary metastasis of tumor cells [4,8,13]. It seems possible that changes in tumor cell surface properties via increased N-glycosylation may be related to the antimetastatic activity of N-1554. Administration of the immunosuppressants carrageenan or anti-aGM, antibody to the host mice abolished the growth inhibitory effect of N-1554 against B16-FlO melanoma. It was suggested that intact function of macrophages and natural killer cells is the prerequisite for the antitumor action of N-1554. In addition, pretreatment of host mice with N-1554 reduced the growth of subcutaneously inoculated B16-FlO melanoma and prolonged the survival time of the tumor-bearing mice. The data suggested that the enhancement of antitumor potential of host immune system may be involved in the effect of N-1554. For further clarifying the mechanism of the antitumor action of N-1554, it may be important to examine the direct effect of N-1554 on the host tumor surveillance system. In conclusion, the present study showed the noteworthy antitumor effect of N-1554 against B16-FlO melanoma. And it was suggested that the effect may be largely mediated by the enhancement of host immune system. References 1
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