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Prostaglandin D2 inhibits the proliferation of human neuroblastoma cells
Cancer Letters, 17 (1983) 289-294 Elsevier Scientific Publishers Ireland Ltd.
289
PROSTAGIANDIN Dz INHIBITS THE PROLIFERATION NEUROBLASTOMA CELLS
TOSHIYUKI SAKAIs, NOZOMI YAMAGUCHI”, NISHINOb and AKIO IWASHIMAb “Department of Preventive Medicine Prefectural University of Medicine,
OF HUMAN
KFXICHI KAWAI*, HOYOKU
and bDepartment of Biochemistry, Kyoto Kawaramachi-Hirokoji, Kamikyoku, Kyoto
602
(Japan) (Received 6 August 1982) (Revised version received 24 September 1982) (Accepted 29 September 1982)
SUMMARY
The cytotoxic action of prostaglandin (PG) D,, E, and F,, was examined on human neuroblastoma cells (NB-1 cell line), and PGD, was found to be the most effective. PGE,, thought to be the most effective among all PGs in the therapy of neuroblastoma, was much less effective than PGD,. PGF,, did not show any inhibitory effect on the proliferation of NB-1 cells. When PGDz was added, the cytoplasma became microscopically larger, then the cells gradually died off. PGD, also exerted a dose-dependent inhibition of DNA and RNA syntheses. These results strongly suggest an antineoplastic activity of PGD? for human neuroblastoma.
INTRODUCTION
Prostaglandin E, has been studied extensively [ 8,14,15,22] for therapy of neuroblastoma. Recently, Dray and Bottenstein reported that PGD, also has a cytotoxic effect on the growth of rat neuroblastoma cells in culture [l].Although the biological function of PGD, is not yet fully understood, it has been described in 3 ways: (1) as an inhibitor of platelet aggregation [WW231; (2)as a neuromodulator [ 191; (3) as a potential antineoplastic agent [ 1,4,9]. In this paper, we present data showing that PGDz has a potent cytotoxic activity on human neuroblastoma cells, and that it is much more cytotoxic than PGE, . The possibility that PGD2 might have a general tumor-growth inhibitory action is also discussed.
0304-3835/83/0000--0000/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
MATERIALS
AND METHODS
Cell culture
In the present study, a human neuroblastoma cell line, NB-1, established by Dr. Miyake in the Department of Pathology of Kyoto Prefectural University of Medicine, was used [ll] . Cells were cultured in a mixed medium of Eagles’s minimum essential medium 45%, RPM1 1640 45%, and fetal bovine serum 10%. PGs were dissolved in 99.5% ethanol and diluted in 2 ml medium in a dish with a final concentration of 0.2% ethanol Control media contained 0.2% ethanol. NB-1 cells were plated at a density of 3 X lo5 cells/2 ml medium in 35-mm diameter dishes, and cultured in a humidified atmosphere of 5% CO, in the air. The next day, the various PGs or ethanol were added. The culture was continued without medium change. After 2,4, and 6 days, the number of viable cells or DNA and RNA syntheses were assayed. Chemicals
Synthetic prostaglandins, PGD2, PGE,, and PGF,, were obtained from Funakoshi Pharmaceutical Co. Ltd. (Tokyo, Japan). The purity of PGD*, PGE, and PGF,, was over 99.9% measured by high pressure liquid chromatography. Fetal bovine serum was purchased from GIBCO. [6-3H]Thymidine (27 Ci/mmol) and [ 5-3H] uridine (27 Ci/mmol) were purchased from Amersham International Ltd. Measurement
of DNA and RNA syntheses
NB-1 cells were incubated for 1 h with 10 pCi of [ 3H] thymidine or 5 PCi of [3H] uridine at 37°C in a humidified atmosphere of 5% CO, in the air. The cells were washed with 2 ml of phosphate buffered saline, 2 ml of 5% trichloroacetic acid (TCA) added, and then kept overnight or longer in a wet chamber. Cells were washed with 2 ml of 5% TCA and solubilized with 0.8 ml of 1% sodium dodecyl sulfate. The radioactivities in aliquots were counted in a liquid scintillation spectrometer. Data are expressed as DPM per mg cellular protein. Protein was determined by a modification of the method of Lowry et al. [lo]. Each experiment was performed in duplicate, using at least 2 different cell cultures. RESULTS
Effect
of PGDz on the proliferation
of NB-1 cells
Figure 1 shows the growth curve of NB-1 cells in the presence or absence of PGD2, PGE1, or PGF2,. PGFza had no effect on the cell growth at 5.0 pg/ml. PGE, decreased the growth rate of NB-1 cells to 64.9% 66.0% and 62.3% of the control level on the 2nd, 4th and 6th day, respectively, after the PG was added. However, the number of cells never decreased but gradually increased with PGE, at 5.0 pg/ml. On the other hand, PGD2
291
Fig. 1. Effects of PGD,, PGE,, and PGF,, on the proliferation of NB-1 cells. NB-1 cells were cultured as described under Materials and Methods. Various kinds of PGs were added on the 1st day after the inoculation of cells. c---o, control; --•, with 5 I.tg/ml of PGD,; a------a, with 5 pg/ml of PGE,; *---A, with 5 gg/ml of PGF,,. The cells in culture dishes were suspended by pipetting and counted with a hemocytometer. Cell viability was measured by trypan blue exclusion. The values &present means of duplicate experiments.
decreased the growth rate of NB-1 cells to 56X%, 24.5% and 4.3% on the 2nd, 4th and 6th day, respectively, after the PG was added. Furthermore, for days 4 and 6, cell numbers were gradually reduced below the level of inoculum at this concentration of PGD,. In other words, there was no cell replication but cell death under these experimental conditions. Microscopically, after the addition of PGD,, the cytoplasm became larger before cell death, However, there was not any manifest sign of cell differentiation, such as the typical axon formation, which we observed after the addition of PGE,. When NB-1 cells were cultured with various concentrations of PGD,
loo!
0.625
125
2.5
5.0
10.0
Concentration of prostaglandinDZ (pg/ml) Fig. 2. Effects of various concentrations of PGD, on the proliferation of NB-1 cells. NB-1 cells were cultured as described under Materials and Methods. Various concentrations of PGD, were added on the 1st day after the inoculation of cells. Culture was continued for 4 days. The values represent means of duplicate experiments.
292
TABLE 1 EFFECT OF PGD, ON DNA AND RNA SYNTHESES OF NB-1 CELLS Culture days
PDG, (ccg/mI) 0
2.5
5.0
15964 48242 47517
9526 15795 30417
2.5
Uridine (dpm/rg
Thymidine (dpm/pg protein)a 3 5 7
0
54 67 53
1500 4561 8877
5.0
protein)a 893 4371 7271
389 80 105
aDNA and RNA syntheses were measured as described under Materials and Methods. The values represent means of duplicate experiments.
for 4 days, a dose-dependent inhibition of growth was observed over 0.625 pg/ml. Furthermore, there was no cell replication but cell death at PGDz concentrations of 5 and 10 pg/ml; IC5,, value was calculated to be 2.5 pg/ml (Fig. 2). Effect
of PGDz on DNA and RNA syntheses
of NB-1 cells
The effects on [3H] thymidine and [3H] uridine incorporation were studied to know the inhibition mechanism of PGD, on cell growth. As shown in Table 1, both DNA and RNA syntheses were suppressed. DNA synthesis was suppressed to 33-64% of the control level with 2.5 pg/ml PGDz. RNA synthesis was suppressed to 60-96% with 2.5 pg/ml. DNA synthesis was suppressed to O.l- 0.3% of the control level with 5 pg/ml PGD?. RNA synthesis was suppressed to 1.2-2677~ with 5 pg/ml. These results show PGDz has a dose-dependent inhibition of DNA and RNA syntheses. DISCUSSION
From the results obtained in this study, we conclude that PGDz inhibits the proliferation of human neuroblastoma cells, and kills those cells. PGE, is known to differentiate neuroblastoma cells. However, PGD, did not cause the manifest sign of cell differentiation, such as the axon formation before cell death. Thus, we conclude that even if cell differentiation occurs, the cytotoxic effect of PGDz is dominant. Although DNA and ,RNA syntheses were inhibited by PGD,, we cannot clearly explain the mechanism of cytotoxicity of PGDz by this evidence only. Further investigation to clarify the mechanism of the cytotoxic action of PGD2 will be carried out. In the preliminary data we found that PGD, also exhibits a cytotoxic effect on GOT0 cells, another human neuroblastoma cell line [ 181. Similar
293
cytotoxic actions of PGDz were observed on human lung, pancreatic, gastric and colonic cancer cells in culture [ 161. Recently, it has been reported that the proliferation of human leukemic cells was inhibited by PGDz [4]. Not only these human tumor cells, but also many animal malignant tumor cells, such as: (1) rat neuroblastoma cells [l] ; (2) mouse malignant mastocytoma cells [ 91; (3) mouse leukemia cells [ 41; (4) simian virus 40-transformed mouse fibroblasts [12], were found to be retarded in growth by treatment with PGD,. And an anti-metastatic action of PGD2 on mouse melanoma cells was also reported [3,21]. Thus, it was presumed that PGDz has a common antineoplastic effect. It is of interest that PGDz caused less cytotoxic effect on normal cells [12] . Among other prostaglandins, PGE and PGA series have been known to inhibit the growth of tumor cells [2,6,7,17]. However, these PGs, particularly PGA?, have stronger catastrophic actions on systemic circulation than PGD2. Therefore, PGDz appears safer to use as an antineoplastic drug than the PGA and PGE series. Since PGDz is known not to be metabolized by NADH-linked 15-hydroxyprostaglandin dehydrogenase, a major PG metabolizing enzyme [ 51, it seems much more advantageous to keep therapeutic concentration in vivo than other PGs. This evidence suggests that PGD2 is a promising drug for malignant tumors. ACKNOWLEDGEMENTS
We thank Dr. M. Sekiguchi in the Department of Clinical Oncology, University of Tokyo for kindly supplying us with NB-1 and GOT0 human neuroblastoma cell lines. Also we thank Dr. S. Imashuku, in the Department of Pediatrics of the Kyoto Prefectural University of Medicine, for his kind advice. REFERENCES 1 Bottenstein, J.E. (1980) Serum-free culture of neuroblastoma cells, In: Progress in Cancer Research and Therapy, 12, Advances in Neuroblastoma Research pp. 161-170. Editor: A.E. Evans. Raven Press, New York. 2 Eisenbarth, G.S., Wellman, D.&and Lebovitz, H.E. (1974) Prostaglandin A, inhibition of chondrosarcoma growth. Biochem. Biophys. Res. Commun., 60,1302-1308. 3 Fitzpatrick, F.A. and Stringfellow, D.A. (1979) Prostaglandin Dz formation by malignant melanoma cells correlates inversely with cellular metastatic potential. Proc. Natl. Acad. Sci. U.S.A., 76, 1765-1769. 4 Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S. and Hayaishi, 0. (1982) Prostaglandin D,, a potential antineoplastic agent. Biochem. Biophys. Res. Commun., 105,956-964. 5 Hansen, H.S. (1976) 15-Hydroxyprostaglandin dehydrogenase. A review. Prostaglandins, 12, 647-679. 6 Honn, K.V., Dunn, II, J.R., Morgan, L.R., Bienkowski, M. and Mamett, L.J. (1979) Inhibition of DNA synthesis in Harding-Passey melanoma cells by prostaglandins A, and A,: Comparison with chemotherapeutic agents. Biochem. Biophys. Res. Commun., 87,795~-801.
294
7 Honn, K.V., Bockman, R.S. and Marnett, L.J. (1981) Prostaglandins and cancer: A review of tumor initiation through tumor metastasis. Prostaglandins, 21, 833-864. 8 Imashuku, S. (1979) In vivo differentiation of neuroblastoma - Review of recent experimental studies. Jpn. J. Pediatr. Surg., 11, 1251-1260. 9 Kawamura, M. and Koshihara, Y. (198 1) Inhibition of the growth of mastocytoma cells by prostaglandin D,. Proc. Jpn. Cancer Assoc., 40, 233 (in Japanese). 10 Markwell, M.A.K., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87, 206-210. 11 Miyake, S., Shimo, Y., Kitamura, T., Nojyo, Y., Nakamura, T., Imashuku, S. and Abe, T. (1973) Characteristics of continuous and functional cell line NB-1, derived from a human neuroblastoma. The Autonomic Nervous System, 10, 115-120. 12 Nishino, H., Sakai, T. and Yamaguchi, N. (1982) The effect of prostaglandin D, on the proliferation of normal and malignant mouse fibroblasts. J. Kyoto Pref. Univ. Med., 91, 669-671. 13 Nishizawa, E.E., Miller, W.L., Gorman, R.R., Bundy, G.L., Svensson, J. and Hamberg, M. (1975) Prostaglandin D, as a potential antithrombotic agent. Prostaglandins, 9, 109-121. 14 Prasad, K.N. (1972) Morphological differentiation induced by prostaglandin in mouse neuroblastoma cells in culture. Nat., New Biol., 236, 49-52. 15 Prasad, K.N. (1975) Differentiation of neuroblastoma cells in culture. Biol. Rev., 50,129-165. 16 Sakai, T., Yamaguchi, N. and Nishino, H. (1982) Prostaglandin D, inhibits the proliferation of various human cancer cell lines. J. Kyoto Pref. Univ. Med., 91, 665--667. 17 Santoro, M.G., Philpott, G.W. and Jaffe, B.M. (1976) Inhibition of tumor growth in vivo and in vitro by prostaglandin E. Nature, 263, 777-779. 18 Sekiguchi, M., Oota, T., Sakakibara, K., Inui, N. and Fujii, G. (1979) Establishment and characterization of a human neuroblastoma cell line in tissue culture. Jpn. J. Exp. Med., 49, 67-83. 19 Shimizu, T., Mizuno, N., Amano, T. and Hayaishi, 0. (1979) Prostaglandin D,, a neuromodulator. Proc. Natl. Acad. Sci. U.S.A., 76, 6231-6234. 20 Smith, J.B., Silver, M.J., Ingerman, C.M. and Kocsis, J.J. (1974) Prostaglandin D, inhibits the aggregation of human platelets. Thromb. Res., 5, 291-299. 21 Stringfellow, D.A. and Fitzpatrick, F.A. (1979) Prostaglandin D, controls pulmonary metastasis of malignant melanoma cells. Nature, 282, 76-78. 22 Todo, S., Nakajima, F., Amano, T., Imashuku, S., Kusunoki, T., Yamasaki, S. and Muneta, S. (1978) Total therapy of a patient with stage IV neuroblastoma. Clinical use of the maturation provocative therapy. J. Jpn. Sot. Pediatr. Surg., 14, 77-85. 23 Whittle, B.J.R., Moncada, S. and Vane, J.R. (1978) Comparison of the effects of prostacyclin (PGI,), prostaglandin E, and D, on platelet aggregation in different species. Prostaglandins, 16, 373-388.
289
PROSTAGIANDIN Dz INHIBITS THE PROLIFERATION NEUROBLASTOMA CELLS
TOSHIYUKI SAKAIs, NOZOMI YAMAGUCHI”, NISHINOb and AKIO IWASHIMAb “Department of Preventive Medicine Prefectural University of Medicine,
OF HUMAN
KFXICHI KAWAI*, HOYOKU
and bDepartment of Biochemistry, Kyoto Kawaramachi-Hirokoji, Kamikyoku, Kyoto
602
(Japan) (Received 6 August 1982) (Revised version received 24 September 1982) (Accepted 29 September 1982)
SUMMARY
The cytotoxic action of prostaglandin (PG) D,, E, and F,, was examined on human neuroblastoma cells (NB-1 cell line), and PGD, was found to be the most effective. PGE,, thought to be the most effective among all PGs in the therapy of neuroblastoma, was much less effective than PGD,. PGF,, did not show any inhibitory effect on the proliferation of NB-1 cells. When PGDz was added, the cytoplasma became microscopically larger, then the cells gradually died off. PGD, also exerted a dose-dependent inhibition of DNA and RNA syntheses. These results strongly suggest an antineoplastic activity of PGD? for human neuroblastoma.
INTRODUCTION
Prostaglandin E, has been studied extensively [ 8,14,15,22] for therapy of neuroblastoma. Recently, Dray and Bottenstein reported that PGD, also has a cytotoxic effect on the growth of rat neuroblastoma cells in culture [l].Although the biological function of PGD, is not yet fully understood, it has been described in 3 ways: (1) as an inhibitor of platelet aggregation [WW231; (2)as a neuromodulator [ 191; (3) as a potential antineoplastic agent [ 1,4,9]. In this paper, we present data showing that PGDz has a potent cytotoxic activity on human neuroblastoma cells, and that it is much more cytotoxic than PGE, . The possibility that PGD2 might have a general tumor-growth inhibitory action is also discussed.
0304-3835/83/0000--0000/$03.00 o 1983 Elsevier Scientific Publishers Ireland Ltd. - Published and Printed in Ireland
MATERIALS
AND METHODS
Cell culture
In the present study, a human neuroblastoma cell line, NB-1, established by Dr. Miyake in the Department of Pathology of Kyoto Prefectural University of Medicine, was used [ll] . Cells were cultured in a mixed medium of Eagles’s minimum essential medium 45%, RPM1 1640 45%, and fetal bovine serum 10%. PGs were dissolved in 99.5% ethanol and diluted in 2 ml medium in a dish with a final concentration of 0.2% ethanol Control media contained 0.2% ethanol. NB-1 cells were plated at a density of 3 X lo5 cells/2 ml medium in 35-mm diameter dishes, and cultured in a humidified atmosphere of 5% CO, in the air. The next day, the various PGs or ethanol were added. The culture was continued without medium change. After 2,4, and 6 days, the number of viable cells or DNA and RNA syntheses were assayed. Chemicals
Synthetic prostaglandins, PGD2, PGE,, and PGF,, were obtained from Funakoshi Pharmaceutical Co. Ltd. (Tokyo, Japan). The purity of PGD*, PGE, and PGF,, was over 99.9% measured by high pressure liquid chromatography. Fetal bovine serum was purchased from GIBCO. [6-3H]Thymidine (27 Ci/mmol) and [ 5-3H] uridine (27 Ci/mmol) were purchased from Amersham International Ltd. Measurement
of DNA and RNA syntheses
NB-1 cells were incubated for 1 h with 10 pCi of [ 3H] thymidine or 5 PCi of [3H] uridine at 37°C in a humidified atmosphere of 5% CO, in the air. The cells were washed with 2 ml of phosphate buffered saline, 2 ml of 5% trichloroacetic acid (TCA) added, and then kept overnight or longer in a wet chamber. Cells were washed with 2 ml of 5% TCA and solubilized with 0.8 ml of 1% sodium dodecyl sulfate. The radioactivities in aliquots were counted in a liquid scintillation spectrometer. Data are expressed as DPM per mg cellular protein. Protein was determined by a modification of the method of Lowry et al. [lo]. Each experiment was performed in duplicate, using at least 2 different cell cultures. RESULTS
Effect
of PGDz on the proliferation
of NB-1 cells
Figure 1 shows the growth curve of NB-1 cells in the presence or absence of PGD2, PGE1, or PGF2,. PGFza had no effect on the cell growth at 5.0 pg/ml. PGE, decreased the growth rate of NB-1 cells to 64.9% 66.0% and 62.3% of the control level on the 2nd, 4th and 6th day, respectively, after the PG was added. However, the number of cells never decreased but gradually increased with PGE, at 5.0 pg/ml. On the other hand, PGD2
291
Fig. 1. Effects of PGD,, PGE,, and PGF,, on the proliferation of NB-1 cells. NB-1 cells were cultured as described under Materials and Methods. Various kinds of PGs were added on the 1st day after the inoculation of cells. c---o, control; --•, with 5 I.tg/ml of PGD,; a------a, with 5 pg/ml of PGE,; *---A, with 5 gg/ml of PGF,,. The cells in culture dishes were suspended by pipetting and counted with a hemocytometer. Cell viability was measured by trypan blue exclusion. The values &present means of duplicate experiments.
decreased the growth rate of NB-1 cells to 56X%, 24.5% and 4.3% on the 2nd, 4th and 6th day, respectively, after the PG was added. Furthermore, for days 4 and 6, cell numbers were gradually reduced below the level of inoculum at this concentration of PGD,. In other words, there was no cell replication but cell death under these experimental conditions. Microscopically, after the addition of PGD,, the cytoplasm became larger before cell death, However, there was not any manifest sign of cell differentiation, such as the typical axon formation, which we observed after the addition of PGE,. When NB-1 cells were cultured with various concentrations of PGD,
loo!
0.625
125
2.5
5.0
10.0
Concentration of prostaglandinDZ (pg/ml) Fig. 2. Effects of various concentrations of PGD, on the proliferation of NB-1 cells. NB-1 cells were cultured as described under Materials and Methods. Various concentrations of PGD, were added on the 1st day after the inoculation of cells. Culture was continued for 4 days. The values represent means of duplicate experiments.
292
TABLE 1 EFFECT OF PGD, ON DNA AND RNA SYNTHESES OF NB-1 CELLS Culture days
PDG, (ccg/mI) 0
2.5
5.0
15964 48242 47517
9526 15795 30417
2.5
Uridine (dpm/rg
Thymidine (dpm/pg protein)a 3 5 7
0
54 67 53
1500 4561 8877
5.0
protein)a 893 4371 7271
389 80 105
aDNA and RNA syntheses were measured as described under Materials and Methods. The values represent means of duplicate experiments.
for 4 days, a dose-dependent inhibition of growth was observed over 0.625 pg/ml. Furthermore, there was no cell replication but cell death at PGDz concentrations of 5 and 10 pg/ml; IC5,, value was calculated to be 2.5 pg/ml (Fig. 2). Effect
of PGDz on DNA and RNA syntheses
of NB-1 cells
The effects on [3H] thymidine and [3H] uridine incorporation were studied to know the inhibition mechanism of PGD, on cell growth. As shown in Table 1, both DNA and RNA syntheses were suppressed. DNA synthesis was suppressed to 33-64% of the control level with 2.5 pg/ml PGDz. RNA synthesis was suppressed to 60-96% with 2.5 pg/ml. DNA synthesis was suppressed to O.l- 0.3% of the control level with 5 pg/ml PGD?. RNA synthesis was suppressed to 1.2-2677~ with 5 pg/ml. These results show PGDz has a dose-dependent inhibition of DNA and RNA syntheses. DISCUSSION
From the results obtained in this study, we conclude that PGDz inhibits the proliferation of human neuroblastoma cells, and kills those cells. PGE, is known to differentiate neuroblastoma cells. However, PGD, did not cause the manifest sign of cell differentiation, such as the axon formation before cell death. Thus, we conclude that even if cell differentiation occurs, the cytotoxic effect of PGDz is dominant. Although DNA and ,RNA syntheses were inhibited by PGD,, we cannot clearly explain the mechanism of cytotoxicity of PGDz by this evidence only. Further investigation to clarify the mechanism of the cytotoxic action of PGD2 will be carried out. In the preliminary data we found that PGD, also exhibits a cytotoxic effect on GOT0 cells, another human neuroblastoma cell line [ 181. Similar
293
cytotoxic actions of PGDz were observed on human lung, pancreatic, gastric and colonic cancer cells in culture [ 161. Recently, it has been reported that the proliferation of human leukemic cells was inhibited by PGDz [4]. Not only these human tumor cells, but also many animal malignant tumor cells, such as: (1) rat neuroblastoma cells [l] ; (2) mouse malignant mastocytoma cells [ 91; (3) mouse leukemia cells [ 41; (4) simian virus 40-transformed mouse fibroblasts [12], were found to be retarded in growth by treatment with PGD,. And an anti-metastatic action of PGD2 on mouse melanoma cells was also reported [3,21]. Thus, it was presumed that PGDz has a common antineoplastic effect. It is of interest that PGDz caused less cytotoxic effect on normal cells [12] . Among other prostaglandins, PGE and PGA series have been known to inhibit the growth of tumor cells [2,6,7,17]. However, these PGs, particularly PGA?, have stronger catastrophic actions on systemic circulation than PGD2. Therefore, PGDz appears safer to use as an antineoplastic drug than the PGA and PGE series. Since PGDz is known not to be metabolized by NADH-linked 15-hydroxyprostaglandin dehydrogenase, a major PG metabolizing enzyme [ 51, it seems much more advantageous to keep therapeutic concentration in vivo than other PGs. This evidence suggests that PGD2 is a promising drug for malignant tumors. ACKNOWLEDGEMENTS
We thank Dr. M. Sekiguchi in the Department of Clinical Oncology, University of Tokyo for kindly supplying us with NB-1 and GOT0 human neuroblastoma cell lines. Also we thank Dr. S. Imashuku, in the Department of Pediatrics of the Kyoto Prefectural University of Medicine, for his kind advice. REFERENCES 1 Bottenstein, J.E. (1980) Serum-free culture of neuroblastoma cells, In: Progress in Cancer Research and Therapy, 12, Advances in Neuroblastoma Research pp. 161-170. Editor: A.E. Evans. Raven Press, New York. 2 Eisenbarth, G.S., Wellman, D.&and Lebovitz, H.E. (1974) Prostaglandin A, inhibition of chondrosarcoma growth. Biochem. Biophys. Res. Commun., 60,1302-1308. 3 Fitzpatrick, F.A. and Stringfellow, D.A. (1979) Prostaglandin Dz formation by malignant melanoma cells correlates inversely with cellular metastatic potential. Proc. Natl. Acad. Sci. U.S.A., 76, 1765-1769. 4 Fukushima, M., Kato, T., Ueda, R., Ota, K., Narumiya, S. and Hayaishi, 0. (1982) Prostaglandin D,, a potential antineoplastic agent. Biochem. Biophys. Res. Commun., 105,956-964. 5 Hansen, H.S. (1976) 15-Hydroxyprostaglandin dehydrogenase. A review. Prostaglandins, 12, 647-679. 6 Honn, K.V., Dunn, II, J.R., Morgan, L.R., Bienkowski, M. and Mamett, L.J. (1979) Inhibition of DNA synthesis in Harding-Passey melanoma cells by prostaglandins A, and A,: Comparison with chemotherapeutic agents. Biochem. Biophys. Res. Commun., 87,795~-801.
294
7 Honn, K.V., Bockman, R.S. and Marnett, L.J. (1981) Prostaglandins and cancer: A review of tumor initiation through tumor metastasis. Prostaglandins, 21, 833-864. 8 Imashuku, S. (1979) In vivo differentiation of neuroblastoma - Review of recent experimental studies. Jpn. J. Pediatr. Surg., 11, 1251-1260. 9 Kawamura, M. and Koshihara, Y. (198 1) Inhibition of the growth of mastocytoma cells by prostaglandin D,. Proc. Jpn. Cancer Assoc., 40, 233 (in Japanese). 10 Markwell, M.A.K., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem., 87, 206-210. 11 Miyake, S., Shimo, Y., Kitamura, T., Nojyo, Y., Nakamura, T., Imashuku, S. and Abe, T. (1973) Characteristics of continuous and functional cell line NB-1, derived from a human neuroblastoma. The Autonomic Nervous System, 10, 115-120. 12 Nishino, H., Sakai, T. and Yamaguchi, N. (1982) The effect of prostaglandin D, on the proliferation of normal and malignant mouse fibroblasts. J. Kyoto Pref. Univ. Med., 91, 669-671. 13 Nishizawa, E.E., Miller, W.L., Gorman, R.R., Bundy, G.L., Svensson, J. and Hamberg, M. (1975) Prostaglandin D, as a potential antithrombotic agent. Prostaglandins, 9, 109-121. 14 Prasad, K.N. (1972) Morphological differentiation induced by prostaglandin in mouse neuroblastoma cells in culture. Nat., New Biol., 236, 49-52. 15 Prasad, K.N. (1975) Differentiation of neuroblastoma cells in culture. Biol. Rev., 50,129-165. 16 Sakai, T., Yamaguchi, N. and Nishino, H. (1982) Prostaglandin D, inhibits the proliferation of various human cancer cell lines. J. Kyoto Pref. Univ. Med., 91, 665--667. 17 Santoro, M.G., Philpott, G.W. and Jaffe, B.M. (1976) Inhibition of tumor growth in vivo and in vitro by prostaglandin E. Nature, 263, 777-779. 18 Sekiguchi, M., Oota, T., Sakakibara, K., Inui, N. and Fujii, G. (1979) Establishment and characterization of a human neuroblastoma cell line in tissue culture. Jpn. J. Exp. Med., 49, 67-83. 19 Shimizu, T., Mizuno, N., Amano, T. and Hayaishi, 0. (1979) Prostaglandin D,, a neuromodulator. Proc. Natl. Acad. Sci. U.S.A., 76, 6231-6234. 20 Smith, J.B., Silver, M.J., Ingerman, C.M. and Kocsis, J.J. (1974) Prostaglandin D, inhibits the aggregation of human platelets. Thromb. Res., 5, 291-299. 21 Stringfellow, D.A. and Fitzpatrick, F.A. (1979) Prostaglandin D, controls pulmonary metastasis of malignant melanoma cells. Nature, 282, 76-78. 22 Todo, S., Nakajima, F., Amano, T., Imashuku, S., Kusunoki, T., Yamasaki, S. and Muneta, S. (1978) Total therapy of a patient with stage IV neuroblastoma. Clinical use of the maturation provocative therapy. J. Jpn. Sot. Pediatr. Surg., 14, 77-85. 23 Whittle, B.J.R., Moncada, S. and Vane, J.R. (1978) Comparison of the effects of prostacyclin (PGI,), prostaglandin E, and D, on platelet aggregation in different species. Prostaglandins, 16, 373-388.