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Antitumor effects of vitamin A and inhibitors of ornithine decarboxylase in cultured neuroblastoma and glioma cells
Life Sciences, Vol. 26, pp. 1359-1366 Printed in the U.S.A.
Pergamon Press
ANTITUMOR EFFECTS OF VITAMIN A AND INHIBITORS OF ORNITHINE DECARBOXYLASE IN CULTUREDNEUROBLASTOMA AND GLIOMA CELLS Sharon K. Chapman Department of Pharmacology and Therapeutics University of Florida Gainesville, Florida 32610 (Received in final form February 20, 1980)
Summary The antiproliferative effects of vitamin A analogs and difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase, were evaluated in cultured neuroblastoma and glioma cells. Retinol and retinoic acid arrested the proliferation of neuroblastoma at concentrations of 50 ~M; retinal was effective at 5 ~M. Glioma cells were 5-I0 fold less sensitive to all three analogs. A correlation existed between the inhibition of growth and the inhibition of ornithine decarboxylase in both cell lines treated with the retinoids. Difluoromethyl ornithine did not toally arrest tumor growth at concentrations of 5 mM; however, the antitumor effects were enhanced with the addition of retinol. An earlier and more complete cytostatic response was observed in glioma cells which had been treated with the combination of lO0 ~M retinol and 5 mMdifluoromethyl ornithine than in cells which had been treated with either agent alone. These results show that tumors of neural origin are retinoid sensitive and suggest that a combination of vitamin A analogs and ornithine decarboxylase inhibitors may produce an increased chemotherapeutic response. Analogs of vitamin A have been used successfully in the prevention of cancer of the skin (I-5), lung (6), bladder (7-9), and breast (lO-12) in experimental animals. Oral or intraperitoneal administration of the vitamin has been shown to inhibit the growth of a transplantable murine melanoma (13) and a mammary adenocarcinoma (14). Although further investigations are needed to establish the therapeutic mechanism, a number of recent studies (15,16) have suggested that retinoids inhibit a sequence of growth events at a common biochemical control point involving the induction of ornithine decarboxylase (ODC). Induction of the activity of this rate-limiting enzyme in polyamine synthesis is an obligatory event in cell growth and proliferation (17,18). Although the molecular mechanism by which vitamin A acts to inhibit ODC is unknown, Haddox and Russell (15) have reported that the inhibitory action is cell cycle dependent. Their studies with Chinese hamster ovary cells demonstrate that vitamin A blocks the mid-Gl induction of ODC and the mechanism appears to involve a block of the transcriptional event required for new enzyme synthesis
(16).
Data from our laboratory have shown that a number of post-transcriptional inhib. itors of ODC produce dramatic decreases in neuroblastoma replication (19,20). Glioma cells were less sensitive in spite of significant drug-induced decreases in enzyme activity. The failure to toally arrest tumor growth was attributed in part to the unusual characteristics of this enzyme. ODC has an extremely short half-life (ll-20 min) (21,22) and continuous synthesis of new enzyme 0024-3205/80/161359-08502.00/0 Copyright (c) 1980 Pergamon Press Ltd
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Vitamin A in Neuroblastoma and Glioma
Vol. 26, No. 16, 1980
during rapid growth may overcome the effects of these inhibitors. An alternative approach is to i n h i b i t the production of the specific mRNAfor the enzyme which was the target site for growth inhibition of CHO cells by vitamin A (16). I f neuroblastoma and glioma cells are retinoid sensitive, then transcriptional inhibition of ODC should offer a therapeutic advantage. In the present work, we accessed the potential antitumor effects of vitamin A analogs and of combinations of vitamin A and an irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO). Methods Materials D,L (l-14C) Ornithine (49.9 mCi/mmole) was obtained from New England Nuclear, Boston, MA. Retinal, retinol, and retinoic acid were purchased from Sigma Chemical Co., St. Louis, MO. Difluoromethyl ornithine was generously donated by the Centre de Recherche Merrell International, Strasbourg (France). Murine C-1300 neuroblastoma cells and rat glioma cells (Clone 6) were obtained from the American Type Culture Collection, Rockville, MD. Modified Eagle medium, Ham's F-lO nutrient medium, fetal bovine serum, and horse serum were purchased from Grand Island Biological Co., Grand Island, NY. Dru9 Treatment Neuroblastoma and glioma cells were maintained as previously described (20) at a constant 37oc with 10% C02-90% a i r and I00% humidity. Difluoromethyl ornithine was dissolved in glass-distilled water, f i l t e r s t e r i l i z e d , and added to the cells in 50 pl quantities to lO ml media such that the final concentration of dimethylsulfoxide was 0.5%. Control cell populations which received 0.5% dimethylsulfoxide showed no alterations in rates of tumor growth. For assays of ornithine decarboxylase a c t i v i t y , l x lO6 cells were seeded in lO0 mm'tissue culture dishes; for cell counting experiments, 5 x I04 cells were seeded in 35 mm tissue culture dishes. Cells were grown for 24 hr after seeding at which time drugs and fresh media were added (time zero). A hemocytometer was used for cell counting; v i a b i l i t y was determined by the a b i l i t y of the cells to exclude trypan blue. Ornithine decarboxylase assays Enzyme assays were performed as previously described (20). Supernatant fractions which had been heated at 65oc for lO min before incubation served as controls. Radioactivity was determined by a Beckman LS 7000 s c i n t i l l a t i o n counter. Protein was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Rockville Centre, NY) using bovine gamma globulin as the standard. Results The cytostatic effects of the naturally occurring retinoids on neuroblastoma are shown in Fig. I. Retinal completely inhibited proliferation at a concentration of 5 ~M. Retinol and retinoic acid were less effective; an arrest of cell doubling occurred at concentrations of 50 IJM. Glioma cells were 5-10 fold less sensitive to all three vitamin A analogs (Fig. 2). Again, retinal was the most potent and arrested cell growth at a concentration of 50 pM. Higher concentrations of retinol and retinoic acid were required (25d ~M) to i n h i b i t early cell doubling.
Vol. 26, No. 16, 198U
Vitamin A in Neuroblastoma and Glioma
1361
When ODC a c t i v i t y was measured, the decrease in enzyme a c t i v i t y correlated with the a n t i p r o l i f e r a t i v e effects of 25 pM and 50 pM retinol in neuroblastoma (Fig. 3). Similarly, the lack of s e n s i t i v i t y of glioma cells to retinol was reflected by the resistance of ODC to inhibition at these concentrations. The best compound available for the depletion of putrescine and spermidine is the irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO) (23,24). However, the enzyme is not completely inhibited at 5 mM DFMOand sufficient putrescine is synthesized to allow normal accumulation of spermine (25-27). A decreased rate of cell proliferation was observed with increasing concentrations of difluoromethyl ornithine in neuroblastoma (Fig. 4), but a significant rate of cell division occurred at a drug concentration of 5 mM. The antitumor effects of combinations of retinol and difluoromethyl ornithine are shown in Fig. 5. In neuroblastoma, the cytostatic effects of 25 uM retinol and 5 mM DFMOwere additive. In contrast, an e a r l i e r and more complete cytostatic response was observed in glioma cells which had been treated with the combination of lO0 pM retinol and 5 mM DFMOthan in cells which had been treated with either agent alone. Cell doubling was completely inhibited within two days after the drug combination was administered while a delay of three days was required before antiproliferative effects could be observed with either agent alone.
z
NEUROBLASTOMA
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Retinol /,o.. / tRetinoic Acid
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DAYS FIG. 1
Effects of retinoids on neuroblastoma cell "division. Key: • , c o n t r o l ; O , retinoid treated. Results are expressed as the ratio N/No where N = number of viable cells per dish at day l , 2, or 3 and No = number of viable cells at time O. Bars represent SEM of 4 to 6 values.
Vitamin A in Neuroblastoma and Glioma
1362
Vol. 26, No. 16, 1980
GLIOMA Z
O
Retinol /i,
Z
Retinoic / Retinal Acid /~0~,
/ / lO,ut
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DAYS FIG. 2 Effects of retinoids on g]ioma cell division. Key: • , control; O , retinoid treated. Bars represent SEM of 4 to 6 values.
GLIOMA
NEUROBLASTOMA
0.8
0.6 _> E
o
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0005 0.2 ¢-
5
0
t 1
I 2
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O
I DAYS
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FIG. 3 Effects of retinol on neuroblastoma and glioma ornithine decarboxylase activity. Key: O , control; • , 5 uM retinol; A , 25 pM r e t i n o l ; [] , 50 pM retinol. Bars represent SEM of 6 to 10 values.
Vol. 26, No. 16, 1980
Vitamin A in Neuroblastoma and Glioma
1363
NEUROBLASTOMA
o
z
I
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DAYS FIG. 4 Effects of difluoromethyl ornithing (DFMO) on neuroblastoma cell division. Key: C) , control; Q , O.l mM DFMO; /~ , l mM DFMO; A , 5 mM DFMO. Bars represent SEM of four values Discussion Previous studies have suggested a correlation between growth rate and ODC activity in cultured glioma and neuroblastoma cells (19,28); continuous polyamine synthesis may be necessary to maintain maximum rates of proliferation. The importance of these cations to cell division would best be demonstrated by producing a state of polyamine depletion; however, a complete block of ODC cannot be demonstrated with direct inhibitors of the enzyme (25). In the present study, only minimal cytostatic effects were observed in neuroblastoma cultures with the irreversible inhibitor, DFMO(Fig. 4). An alternative approach to the direct inhibition of ODC is to inhibit the production of the specific mRNAfor the enzyme. This transcriptional event has been described as a target site for growth inhibition of CHO cells by vitamin A (16). In the present report, the proliferation of cultured neuroblastoma and glioma cells were markedly inhibited by retinol, retinoic acid, and retinal (Figs. l and 2). Although more work is needed to establish the mechanism of the antitumor effects, a correlation was shown between the ability of retinol
1364
Vitamin A in Neuroblastoma and Glioma
GLIOMA
NEUROBLASTOMA
9
,oo~. 75
o
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Vol. 26, No. 16, 1980
I
0 ¢.y
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FIG. 5 Effects of retinol and difluoromethyl ornithine (DFMO) on neuroblastoma and glioma cell division. Neuroblastoma key: 0 , control; Q , 25 ~M retinol; [-1, 5 mM DFMO; • , 50 ~M retinol; / ~ , 25 uM retinol plus 5 mM DFMO; A , 50 pM retinol plus 5 mM DFMO. Glioma key: 0 , control; • , 50 uM retinol; • , lO0 ~M retinol; I-I, 5 mM DFMO; / ~ , lO0 pM retinol plus 5 mM DFMO. Bars represent SEM of 4 to 6 values.
to i n h i b i t ODC and i t s potency as an i n h i b i t o r of p r o l i f e r a t i o n . At a concentration of 50 ~M, retinol arrested neuroblastoma tumor growth (Fig. I) and ODC a c t i v i t y was reduced by 95% in 24 hr (Fig. 3). In contrast, glioma cells were r e l a t i v e l y insensitive to 50 pM retinol (Fig. 2) and a lag of at least two days was required before an a n t i p r o l i f e r a t i v e effect was observed; glioma ODC a c t i v i t y was only s l i g h t l y inhibited at this concentration (36% i n h i b i t i o n at 24 hrs) (Fig. 3). Although our results suggest a relationship between the effects of retinol on growth and on ODC a c t i v i t y , further studies would be required to establish ODC mRNA production as the antitumor mechanism. Other investigators have suggested that the antitumor action of vitamin A is mediated through an immune mechanism (13), through lysosomal labilization (29,30), through an inhibition of choline incorporation into phospholipids (31), through alterations in prostaglandin synthesis ( l ) , and through restoration of contact inhibition (32). In view of these reports i t is possible that multiple sites for retinoid action exist and different mechanisms may be expressed in different cell types (16). A correlation between the a b i l i t y of retinoids to i n h i b i t ODC induction and their potencies as inhibitors of epidermal tumor promotion has been demonstrated (1,2,4,5).
Vol. 26, No. 16, 1980
Vitamin A in Neuroblastoma and Glioma
1365
In the present study, we accessed the effects of combinations of vitamin A and DFMO. I f the two drugs act at different steps in the induction of the enzyme (e.g. as an inhibitor of ODC mRNAproduction and as a direct inhibitor of the enzyme), the combination would be expected to yield at least additive effectiveness. In neuroblastoma studies, the antiproliferative effects of combinations of retinol and DFMOwere additive (Fig. 5); however, in glioma cells, an earlier and more complete cytostatic response was observed with the combination than was observed with either agent alone (Fig. 5). Since the combination produced additive inhibition of ODC in both cell lines (results not shown), i t is possible that there are multiple retinoid sites for growth inhibition in glioma cells. Among the problems associated with the use of retinoids, are the toxic effects which hinder utilization of a completely effective dose (3,33). The results of our studies suggest that, in combination with ODC inhibitors, lower doses of vitamin A might be administered with increased inhibitory responses. We are currently exploring the chemotherapeutic effectiveness of this combination in the neuroblastoma animal model. Acknowledgements The author wishes to thank Ms. Judith Breiner and Ms. Susan Giant for their technical assistance. References 1. 2. 3. 4. 5. 6. 7. 8. 9. lO. II. 12. 13. 14. 15. 16. 17. 18. 19.
A.K. VERMAand R.K. BOUTWELL, Cancer Res. 37, 2196-2201 (1977). A.K. VERMA, J.W. HOLDER, and R.K. BOUTWELL, Proc. Am. Assoc. Cancer Res. 18, 14 (1977). H. MAYER, W. BOLLAG, R. HANNI, and R. RUEGG, Experentia 34, If05-1119 (1978) A.K. VERMA, H.M. RICE, B.G. SHAPAS, and R.K. BOUTWELL, Ca-n-cer Res. 38, 793-801 (1978). A.K. VERMA, B.G. SHAPAS, H.M. RICE, and R.K. BOUTWELL, Cancer Res. 39, 419-425 (1979). P. NETTESHEIMand M.L. WILLIAMS, Int. J. Cancer 17, 351-357 (1976). M.B. SPORN, R.A. SQUIRE, C.C. BROWN, J.M. SMITH,-M.L. WENK, and S. SPRINGER, Science 195, 487-489 (1977). C.J. GRUBBS, R.C. MOON, R.A. SQUIRE, G.M. FARROW, S.F. STINSON, D.G. GOODMAN, C.C. BROWN, and M.B. SPORN, Science 198, 743-744 (1977). P.J. BECCI, H.J. THOMPSON,C.J. GRUBBS, R.A. SQUIRE, C.C. BROWN,M.B. SPORN, and R.C. MOON, Cancer Res. 38, 4463-4466 (1978). R.C. MOON, C.J. GRUBBS, M.B. SPORN, and D.G. GOODMAN,Nature (London) 267, 620-621 (1977). C.J. GRUBBS, R.C. MOON,M.B. SPORNand D.L. NEWTON, Cancer Res. 37, 599602 (1977). R.C. MOON, H.J. THOMPSON, P.J. BECCI, C.J. GRUBBS, R.J. GANDER, D.L. NEWTON, J.M. SMITH, S.L. PHILLIPS, W.R. HENDERSON, L.T. MULLEN, C.C. BROWN, and M.B. SPORN, Cancer Res. 39, 1339-1346 (1979). E.L. FELIX, B. LOYD, and M.H.---COHEN, Science 189, 886 (1975). G. RETTURA,A. SCHITTEK, M. HARDY, S.M. LEVENSON, A. DEMETRIOU, and E. SEIFTER, J. Natl. Cancer Inst. 54, 1489-1491 (1975). M.K. HADDOXand D.H. RUSSELL, Cancer Res. 39, 2476-2480 (1979). M.K. HADDOX, K.F. SCOTT, and D.H. RUSSELL, Cancer Res. 39, 4930-4938 (1979). J. JANNE, H. POSO, and R. RAINA, Biochim. Biophys. Acta 473, 241-293 (1978). D.H. RUSSELLand B.G.M. DURIE, Polyamines as Biochemical Markers of Normal and Malignant Growth, Raven Press, New York (1978). S.K. CHAPMAN, R.L. HAWKE, S.K. GLANT, S.S. MANN, K.S. HWANG, and J.W. DOYLE, Pharmacologist 21, 233 (1979).
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20. 21. 22. 23. 24. 25, 26. 27. 28. 29. 30. 31. 32. 33.
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Vol. 26, No. 16, 1980
S.K. CHAPMAN, M. MARTIN, M.S. HOOVER, and C.Y. CHIOU, Biochem. Pharmacol. 27, 717-721 (1978). D.H. RUSSELL and S.H. SNYDER, Molec. Pharmacol. 5, 253-262 (1962). J.E. KAY, V.J. LINDSAY, and A. COOKE, FEBS Lett. 21, 123-126 (1972). B.W. METCALF, P. BEY, C. DANZlN, M.J. JUNG, P. CASARA, and J.P. VEVERT, J. Amer. Chem. Soc. I00, 2551-2553 (1978). P. BEY and J.P. VEVERT, Tet. Letters 14, 1215-1218 (1978). P.S. MAMONT,M.C. DUCHESNE,A.J. JODER-OHLENBUSCHand J. GROVE, EnzymeActivated Irreversible I n h i b i t o r s , (Eds. N. Seiler, M.J. Jung, and J. KochWeser) Elsevier/North-Holland Biomedical Press (1978). P.S. MAMONT,M. DUCHESNE, J. GROVE, and P. BEY, Biochem. Biophys. Res. Commun. 81, 58-66 (1978). C. DANZIN~M. JUNG, J. GROVE, and P. BEY, Life Sci. 24, 519-524 (1979). U. BACHRACH, FEBS Lett. 68, 63-67 (1976). J.T. DINGLE, Lysosomes, Ci-ba Foundation Symposium (Eds. A.V.S. de Reuck, M.P. Cameron) Boston, L i t t l e , Brown and Company, pp. 384-398 (1963). D. BRANDES, E. ANTON, B. SCHOFIELD and S.R. BARNARD, Cancer Chemother. Rept. 50, 47-53 (1966). P.W. WER---TZand G.C. MUELLER, Cancer Res. 38, 2900-2904 (1978). L.D. DION, J.E. BLALOCK, and G.E. GIFFORD, J. Natl. Cancer Inst. 58, 795-801 (1977). M.D. SPORN, Nutrition Rev. 35, 65-69 (1977).
Pergamon Press
ANTITUMOR EFFECTS OF VITAMIN A AND INHIBITORS OF ORNITHINE DECARBOXYLASE IN CULTUREDNEUROBLASTOMA AND GLIOMA CELLS Sharon K. Chapman Department of Pharmacology and Therapeutics University of Florida Gainesville, Florida 32610 (Received in final form February 20, 1980)
Summary The antiproliferative effects of vitamin A analogs and difluoromethyl ornithine, an irreversible inhibitor of ornithine decarboxylase, were evaluated in cultured neuroblastoma and glioma cells. Retinol and retinoic acid arrested the proliferation of neuroblastoma at concentrations of 50 ~M; retinal was effective at 5 ~M. Glioma cells were 5-I0 fold less sensitive to all three analogs. A correlation existed between the inhibition of growth and the inhibition of ornithine decarboxylase in both cell lines treated with the retinoids. Difluoromethyl ornithine did not toally arrest tumor growth at concentrations of 5 mM; however, the antitumor effects were enhanced with the addition of retinol. An earlier and more complete cytostatic response was observed in glioma cells which had been treated with the combination of lO0 ~M retinol and 5 mMdifluoromethyl ornithine than in cells which had been treated with either agent alone. These results show that tumors of neural origin are retinoid sensitive and suggest that a combination of vitamin A analogs and ornithine decarboxylase inhibitors may produce an increased chemotherapeutic response. Analogs of vitamin A have been used successfully in the prevention of cancer of the skin (I-5), lung (6), bladder (7-9), and breast (lO-12) in experimental animals. Oral or intraperitoneal administration of the vitamin has been shown to inhibit the growth of a transplantable murine melanoma (13) and a mammary adenocarcinoma (14). Although further investigations are needed to establish the therapeutic mechanism, a number of recent studies (15,16) have suggested that retinoids inhibit a sequence of growth events at a common biochemical control point involving the induction of ornithine decarboxylase (ODC). Induction of the activity of this rate-limiting enzyme in polyamine synthesis is an obligatory event in cell growth and proliferation (17,18). Although the molecular mechanism by which vitamin A acts to inhibit ODC is unknown, Haddox and Russell (15) have reported that the inhibitory action is cell cycle dependent. Their studies with Chinese hamster ovary cells demonstrate that vitamin A blocks the mid-Gl induction of ODC and the mechanism appears to involve a block of the transcriptional event required for new enzyme synthesis
(16).
Data from our laboratory have shown that a number of post-transcriptional inhib. itors of ODC produce dramatic decreases in neuroblastoma replication (19,20). Glioma cells were less sensitive in spite of significant drug-induced decreases in enzyme activity. The failure to toally arrest tumor growth was attributed in part to the unusual characteristics of this enzyme. ODC has an extremely short half-life (ll-20 min) (21,22) and continuous synthesis of new enzyme 0024-3205/80/161359-08502.00/0 Copyright (c) 1980 Pergamon Press Ltd
1360
Vitamin A in Neuroblastoma and Glioma
Vol. 26, No. 16, 1980
during rapid growth may overcome the effects of these inhibitors. An alternative approach is to i n h i b i t the production of the specific mRNAfor the enzyme which was the target site for growth inhibition of CHO cells by vitamin A (16). I f neuroblastoma and glioma cells are retinoid sensitive, then transcriptional inhibition of ODC should offer a therapeutic advantage. In the present work, we accessed the potential antitumor effects of vitamin A analogs and of combinations of vitamin A and an irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO). Methods Materials D,L (l-14C) Ornithine (49.9 mCi/mmole) was obtained from New England Nuclear, Boston, MA. Retinal, retinol, and retinoic acid were purchased from Sigma Chemical Co., St. Louis, MO. Difluoromethyl ornithine was generously donated by the Centre de Recherche Merrell International, Strasbourg (France). Murine C-1300 neuroblastoma cells and rat glioma cells (Clone 6) were obtained from the American Type Culture Collection, Rockville, MD. Modified Eagle medium, Ham's F-lO nutrient medium, fetal bovine serum, and horse serum were purchased from Grand Island Biological Co., Grand Island, NY. Dru9 Treatment Neuroblastoma and glioma cells were maintained as previously described (20) at a constant 37oc with 10% C02-90% a i r and I00% humidity. Difluoromethyl ornithine was dissolved in glass-distilled water, f i l t e r s t e r i l i z e d , and added to the cells in 50 pl quantities to lO ml media such that the final concentration of dimethylsulfoxide was 0.5%. Control cell populations which received 0.5% dimethylsulfoxide showed no alterations in rates of tumor growth. For assays of ornithine decarboxylase a c t i v i t y , l x lO6 cells were seeded in lO0 mm'tissue culture dishes; for cell counting experiments, 5 x I04 cells were seeded in 35 mm tissue culture dishes. Cells were grown for 24 hr after seeding at which time drugs and fresh media were added (time zero). A hemocytometer was used for cell counting; v i a b i l i t y was determined by the a b i l i t y of the cells to exclude trypan blue. Ornithine decarboxylase assays Enzyme assays were performed as previously described (20). Supernatant fractions which had been heated at 65oc for lO min before incubation served as controls. Radioactivity was determined by a Beckman LS 7000 s c i n t i l l a t i o n counter. Protein was determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Rockville Centre, NY) using bovine gamma globulin as the standard. Results The cytostatic effects of the naturally occurring retinoids on neuroblastoma are shown in Fig. I. Retinal completely inhibited proliferation at a concentration of 5 ~M. Retinol and retinoic acid were less effective; an arrest of cell doubling occurred at concentrations of 50 IJM. Glioma cells were 5-10 fold less sensitive to all three vitamin A analogs (Fig. 2). Again, retinal was the most potent and arrested cell growth at a concentration of 50 pM. Higher concentrations of retinol and retinoic acid were required (25d ~M) to i n h i b i t early cell doubling.
Vol. 26, No. 16, 198U
Vitamin A in Neuroblastoma and Glioma
1361
When ODC a c t i v i t y was measured, the decrease in enzyme a c t i v i t y correlated with the a n t i p r o l i f e r a t i v e effects of 25 pM and 50 pM retinol in neuroblastoma (Fig. 3). Similarly, the lack of s e n s i t i v i t y of glioma cells to retinol was reflected by the resistance of ODC to inhibition at these concentrations. The best compound available for the depletion of putrescine and spermidine is the irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO) (23,24). However, the enzyme is not completely inhibited at 5 mM DFMOand sufficient putrescine is synthesized to allow normal accumulation of spermine (25-27). A decreased rate of cell proliferation was observed with increasing concentrations of difluoromethyl ornithine in neuroblastoma (Fig. 4), but a significant rate of cell division occurred at a drug concentration of 5 mM. The antitumor effects of combinations of retinol and difluoromethyl ornithine are shown in Fig. 5. In neuroblastoma, the cytostatic effects of 25 uM retinol and 5 mM DFMOwere additive. In contrast, an e a r l i e r and more complete cytostatic response was observed in glioma cells which had been treated with the combination of lO0 pM retinol and 5 mM DFMOthan in cells which had been treated with either agent alone. Cell doubling was completely inhibited within two days after the drug combination was administered while a delay of three days was required before antiproliferative effects could be observed with either agent alone.
z
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O
O
z
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Retinol /,o.. / tRetinoic Acid
O
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2
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DAYS FIG. 1
Effects of retinoids on neuroblastoma cell "division. Key: • , c o n t r o l ; O , retinoid treated. Results are expressed as the ratio N/No where N = number of viable cells per dish at day l , 2, or 3 and No = number of viable cells at time O. Bars represent SEM of 4 to 6 values.
Vitamin A in Neuroblastoma and Glioma
1362
Vol. 26, No. 16, 1980
GLIOMA Z
O
Retinol /i,
Z
Retinoic / Retinal Acid /~0~,
/ / lO,ut
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DAYS FIG. 2 Effects of retinoids on g]ioma cell division. Key: • , control; O , retinoid treated. Bars represent SEM of 4 to 6 values.
GLIOMA
NEUROBLASTOMA
0.8
0.6 _> E
o
~_
0.4
0005 0.2 ¢-
5
0
t 1
I 2
DAYS
O
I DAYS
2
FIG. 3 Effects of retinol on neuroblastoma and glioma ornithine decarboxylase activity. Key: O , control; • , 5 uM retinol; A , 25 pM r e t i n o l ; [] , 50 pM retinol. Bars represent SEM of 6 to 10 values.
Vol. 26, No. 16, 1980
Vitamin A in Neuroblastoma and Glioma
1363
NEUROBLASTOMA
o
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DAYS FIG. 4 Effects of difluoromethyl ornithing (DFMO) on neuroblastoma cell division. Key: C) , control; Q , O.l mM DFMO; /~ , l mM DFMO; A , 5 mM DFMO. Bars represent SEM of four values Discussion Previous studies have suggested a correlation between growth rate and ODC activity in cultured glioma and neuroblastoma cells (19,28); continuous polyamine synthesis may be necessary to maintain maximum rates of proliferation. The importance of these cations to cell division would best be demonstrated by producing a state of polyamine depletion; however, a complete block of ODC cannot be demonstrated with direct inhibitors of the enzyme (25). In the present study, only minimal cytostatic effects were observed in neuroblastoma cultures with the irreversible inhibitor, DFMO(Fig. 4). An alternative approach to the direct inhibition of ODC is to inhibit the production of the specific mRNAfor the enzyme. This transcriptional event has been described as a target site for growth inhibition of CHO cells by vitamin A (16). In the present report, the proliferation of cultured neuroblastoma and glioma cells were markedly inhibited by retinol, retinoic acid, and retinal (Figs. l and 2). Although more work is needed to establish the mechanism of the antitumor effects, a correlation was shown between the ability of retinol
1364
Vitamin A in Neuroblastoma and Glioma
GLIOMA
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FIG. 5 Effects of retinol and difluoromethyl ornithine (DFMO) on neuroblastoma and glioma cell division. Neuroblastoma key: 0 , control; Q , 25 ~M retinol; [-1, 5 mM DFMO; • , 50 ~M retinol; / ~ , 25 uM retinol plus 5 mM DFMO; A , 50 pM retinol plus 5 mM DFMO. Glioma key: 0 , control; • , 50 uM retinol; • , lO0 ~M retinol; I-I, 5 mM DFMO; / ~ , lO0 pM retinol plus 5 mM DFMO. Bars represent SEM of 4 to 6 values.
to i n h i b i t ODC and i t s potency as an i n h i b i t o r of p r o l i f e r a t i o n . At a concentration of 50 ~M, retinol arrested neuroblastoma tumor growth (Fig. I) and ODC a c t i v i t y was reduced by 95% in 24 hr (Fig. 3). In contrast, glioma cells were r e l a t i v e l y insensitive to 50 pM retinol (Fig. 2) and a lag of at least two days was required before an a n t i p r o l i f e r a t i v e effect was observed; glioma ODC a c t i v i t y was only s l i g h t l y inhibited at this concentration (36% i n h i b i t i o n at 24 hrs) (Fig. 3). Although our results suggest a relationship between the effects of retinol on growth and on ODC a c t i v i t y , further studies would be required to establish ODC mRNA production as the antitumor mechanism. Other investigators have suggested that the antitumor action of vitamin A is mediated through an immune mechanism (13), through lysosomal labilization (29,30), through an inhibition of choline incorporation into phospholipids (31), through alterations in prostaglandin synthesis ( l ) , and through restoration of contact inhibition (32). In view of these reports i t is possible that multiple sites for retinoid action exist and different mechanisms may be expressed in different cell types (16). A correlation between the a b i l i t y of retinoids to i n h i b i t ODC induction and their potencies as inhibitors of epidermal tumor promotion has been demonstrated (1,2,4,5).
Vol. 26, No. 16, 1980
Vitamin A in Neuroblastoma and Glioma
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In the present study, we accessed the effects of combinations of vitamin A and DFMO. I f the two drugs act at different steps in the induction of the enzyme (e.g. as an inhibitor of ODC mRNAproduction and as a direct inhibitor of the enzyme), the combination would be expected to yield at least additive effectiveness. In neuroblastoma studies, the antiproliferative effects of combinations of retinol and DFMOwere additive (Fig. 5); however, in glioma cells, an earlier and more complete cytostatic response was observed with the combination than was observed with either agent alone (Fig. 5). Since the combination produced additive inhibition of ODC in both cell lines (results not shown), i t is possible that there are multiple retinoid sites for growth inhibition in glioma cells. Among the problems associated with the use of retinoids, are the toxic effects which hinder utilization of a completely effective dose (3,33). The results of our studies suggest that, in combination with ODC inhibitors, lower doses of vitamin A might be administered with increased inhibitory responses. We are currently exploring the chemotherapeutic effectiveness of this combination in the neuroblastoma animal model. Acknowledgements The author wishes to thank Ms. Judith Breiner and Ms. Susan Giant for their technical assistance. References 1. 2. 3. 4. 5. 6. 7. 8. 9. lO. II. 12. 13. 14. 15. 16. 17. 18. 19.
A.K. VERMAand R.K. BOUTWELL, Cancer Res. 37, 2196-2201 (1977). A.K. VERMA, J.W. HOLDER, and R.K. BOUTWELL, Proc. Am. Assoc. Cancer Res. 18, 14 (1977). H. MAYER, W. BOLLAG, R. HANNI, and R. RUEGG, Experentia 34, If05-1119 (1978) A.K. VERMA, H.M. RICE, B.G. SHAPAS, and R.K. BOUTWELL, Ca-n-cer Res. 38, 793-801 (1978). A.K. VERMA, B.G. SHAPAS, H.M. RICE, and R.K. BOUTWELL, Cancer Res. 39, 419-425 (1979). P. NETTESHEIMand M.L. WILLIAMS, Int. J. Cancer 17, 351-357 (1976). M.B. SPORN, R.A. SQUIRE, C.C. BROWN, J.M. SMITH,-M.L. WENK, and S. SPRINGER, Science 195, 487-489 (1977). C.J. GRUBBS, R.C. MOON, R.A. SQUIRE, G.M. FARROW, S.F. STINSON, D.G. GOODMAN, C.C. BROWN, and M.B. SPORN, Science 198, 743-744 (1977). P.J. BECCI, H.J. THOMPSON,C.J. GRUBBS, R.A. SQUIRE, C.C. BROWN,M.B. SPORN, and R.C. MOON, Cancer Res. 38, 4463-4466 (1978). R.C. MOON, C.J. GRUBBS, M.B. SPORN, and D.G. GOODMAN,Nature (London) 267, 620-621 (1977). C.J. GRUBBS, R.C. MOON,M.B. SPORNand D.L. NEWTON, Cancer Res. 37, 599602 (1977). R.C. MOON, H.J. THOMPSON, P.J. BECCI, C.J. GRUBBS, R.J. GANDER, D.L. NEWTON, J.M. SMITH, S.L. PHILLIPS, W.R. HENDERSON, L.T. MULLEN, C.C. BROWN, and M.B. SPORN, Cancer Res. 39, 1339-1346 (1979). E.L. FELIX, B. LOYD, and M.H.---COHEN, Science 189, 886 (1975). G. RETTURA,A. SCHITTEK, M. HARDY, S.M. LEVENSON, A. DEMETRIOU, and E. SEIFTER, J. Natl. Cancer Inst. 54, 1489-1491 (1975). M.K. HADDOXand D.H. RUSSELL, Cancer Res. 39, 2476-2480 (1979). M.K. HADDOX, K.F. SCOTT, and D.H. RUSSELL, Cancer Res. 39, 4930-4938 (1979). J. JANNE, H. POSO, and R. RAINA, Biochim. Biophys. Acta 473, 241-293 (1978). D.H. RUSSELLand B.G.M. DURIE, Polyamines as Biochemical Markers of Normal and Malignant Growth, Raven Press, New York (1978). S.K. CHAPMAN, R.L. HAWKE, S.K. GLANT, S.S. MANN, K.S. HWANG, and J.W. DOYLE, Pharmacologist 21, 233 (1979).
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S.K. CHAPMAN, M. MARTIN, M.S. HOOVER, and C.Y. CHIOU, Biochem. Pharmacol. 27, 717-721 (1978). D.H. RUSSELL and S.H. SNYDER, Molec. Pharmacol. 5, 253-262 (1962). J.E. KAY, V.J. LINDSAY, and A. COOKE, FEBS Lett. 21, 123-126 (1972). B.W. METCALF, P. BEY, C. DANZlN, M.J. JUNG, P. CASARA, and J.P. VEVERT, J. Amer. Chem. Soc. I00, 2551-2553 (1978). P. BEY and J.P. VEVERT, Tet. Letters 14, 1215-1218 (1978). P.S. MAMONT,M.C. DUCHESNE,A.J. JODER-OHLENBUSCHand J. GROVE, EnzymeActivated Irreversible I n h i b i t o r s , (Eds. N. Seiler, M.J. Jung, and J. KochWeser) Elsevier/North-Holland Biomedical Press (1978). P.S. MAMONT,M. DUCHESNE, J. GROVE, and P. BEY, Biochem. Biophys. Res. Commun. 81, 58-66 (1978). C. DANZIN~M. JUNG, J. GROVE, and P. BEY, Life Sci. 24, 519-524 (1979). U. BACHRACH, FEBS Lett. 68, 63-67 (1976). J.T. DINGLE, Lysosomes, Ci-ba Foundation Symposium (Eds. A.V.S. de Reuck, M.P. Cameron) Boston, L i t t l e , Brown and Company, pp. 384-398 (1963). D. BRANDES, E. ANTON, B. SCHOFIELD and S.R. BARNARD, Cancer Chemother. Rept. 50, 47-53 (1966). P.W. WER---TZand G.C. MUELLER, Cancer Res. 38, 2900-2904 (1978). L.D. DION, J.E. BLALOCK, and G.E. GIFFORD, J. Natl. Cancer Inst. 58, 795-801 (1977). M.D. SPORN, Nutrition Rev. 35, 65-69 (1977).