[CANCER RESEARCH 45, 4067-4070, September 1985]

Effect of Inhibition of Polyamine Biosynthesis by DL-a-Difluoromethylornithine on the Growth and Melanogenesis of B16 Melanoma in Vitro and in Vivo

Prasad S. Sunkara,1 C. C. Chang, N. J. Prakash, and P. J. Lachmann

Merrell Dow Research Institute, Cincinnati, Ohio 45215

ABSTRACT seems to vary with the model systems studied. The studies by Oka ef al. (13) have shown that spermidine is an essential The objective of the present study was to investigate the effect requirement for milk protein synthesis, a differentiated function of polyamine depletion by a-difluoromethylornithine (DFMO), a in mammary glands. Casein and a-lactalbumin synthesis can be specific irreversible inhibitor of ornithine decarboxylase, on the blocked by inhibiting spermidine synthesis. These authors have growth and differentiation of B16 melanoma cells grown in also shown that increased synthesis of putrescine is needed for culture and also as solid tumors in mice. Polyamine depletion by mammary cell growth and that spermidine is essential for milk DFMO (2.5 HIM) resulted in a complete inhibition of cell growth protein production. in culture and a 90% inhibition of viability of melanoma cells as Friend erythroleukemia cells in culture can be induced to determined by clonogenic assay at the end of 7 days after DFMO differentiate by compounds such as dimethyl sulfoxide and hex- treatment. These results indicate that polyamine depletion in amethylene bisacetamide. These agents were shown to enhance duced by DFMO is cytotoxic to B16 melanoma cells in culture. polyamine synthesis. Inhibitors of polyamine biosynthesis pre Furthermore a 2- to 5-fold increase in tyrosinase activity and 10- vented the differentiation stimulated by the inducers and this fold accumulation of melanine were observed in polyamine de inhibition could be reversed by exogenously added polyamines pleted cells compared to control cultures. These effects of DFMO suggesting a possible role for polyamines in Friend erythroleu could easily be reversed by the addition of putrescine simulta kemia cell differentiation (14). Similar increases in polyamine neously with DFMO. Administration of different doses of DFMO levels were observed by agents inducing terminal differentiation in drinking water to B16 melanoma tumor bearing mice also in human HL-60 promyelocytic leukemia cells. However, induc resulted in an increase in tyrosinase activity and a dose depend tion of differentiation in HL-60 cells was not affected by inhibitors ent inhibition (86-90%) of tumor growth. Although one cannot of polyamine synthesis (15,16). rule out the possibility of induction of differentiated phenotype Verma and Sunkara (17) reported an enhanced polyamine as a result of antiproliferative activity of DFMO, the data pre synthesis during colony-stimulating factor-induced proliferation sented indicate that the unique sensitivity of melanoma to DFMO and differentiation of human granulocyte-macrophage progenitor may be due to a combination of cell growth inhibition and cells. Furthermore inhibition of polyamine biosynthesis by DFMO concomitant induction of differentiation upon polyamine deple resulted in an inhibition of differentiation that could be reversed tion. The results of the present study indicate that polyamines easily by an exogenous supply of putrescine suggesting an play an important role in growth and differentiation of melanoma essential role for polyamines during granulopoietic differentiation. and also provide an example of inhibition of tumor cell growth Requirement for polyamines during differentiation has been re by induction of cellular differentiation. ported in a number of model systems. For example matrix- induced endochondrial bone differentiation (18), differentiation of INTRODUCTION rabbit costal chondrocytes (19), differentiation of 3T3-L1 fibro- blasts into adipocytes (20), and differentiation of L6 myoblasts The naturally occurring polyamines, putrescine, spermidine, into myotubes (21). and spermine play an important role in growth and differentiation In contrast to the above studies, in certain model systems of mammalian cells (1-3). The availability of DFMO2, an irrever inhibition of polyamine synthesis was shown to induce differen sible inhibitor of ornithine decarboxylase (4), a rate limiting en tiation. Chen ef a/. (22) have reported that cyclic AMP-induced zyme in the polyamine biosynthetic pathway made it possible to neuroblastoma cell differentiation is accompanied by a decrease understand the role of polyamines in proliferation and differentia in ornithine decarboxylase and polyamines. Furthermore they tion of mammalian cells. DFMO causes a rapid depletion of have also shown that DFMO potentiates the neuroblastoma cell intracellular putrescine and spermidine levels and inhibits growth differentiation at suboptimal concentrations of dibutyryl-cyclic of a variety of cells in culture (5-8). This growth inhibition was AMP. DFMO also stimulates erythroid differentiation of hemato- attributed to the cytostatic nature of DFMO except in the case poietic precursor cells (23) and promotes the differentiation of of small cell lung cancer cells in culture where it was shown to malignant teratocarcinoma cells (24). In the present study we be cytotoxic (5). Inhibition of polyamine biosynthesis by DFMO have investigated the effect of inhibition of polyamine biosyn has been shown to retard tumor growth and metastasis in thesis by DFMO on the growth and differentiation of B16 mela several transplanted animal tumors (9-12). noma cells grown in cell cultures and also as solid tumors in Although the requirement of polyamines for cell growth is quite mice. The results of our studies indicate that polyamine depletion clear the role for these polycations in cellular differentiation causes inhibition of cell growth and enhances melanogenesis as

1To whom requests for reprints should be addressed. indicated by a stimulation of tyrosinase and accumulation of 2The abbreviation used is: DFMO, a-difluoromethylornithine. melanin both in vitro and in vivo. An abstract of this study Received 1/24/85; revised 5/17/85; accepted 6/7/85. appeared elsewhere (25). Similar enhancement of melanogenesis

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4067

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. INHIBITION OF POLY AMINE BIOSYNTHESIS BY DFMO ON B16 MELANOMA in response to DFMO in Cloudman S91 melanoma cells in culture mice. Rapidly dividing 2-week-old tumors were excised from the animals was reported by Kapyaho and Janne (26). and trypsinized. The resulting cell suspension was passed through sterile gauze. The viability of the tumor cells in the supernatant was determined by trypan blue dye exclusion method. For routine maintenance, 1 x 105 MATERIALS AND METHODS viable B16 melanoma cells were injected s.c. at the interscapular region. Chemicals. DFMO was synthesized as the hydrochloride monohy- Antitumor Activity. Male C57BL mice, initial body weight 18-20 g, drate salt (DL-a-difluoromethylornithine •HCI•H2O)at the Merrell Dow obtained from Jackson Laboratories, Bar Harbor, ME, were used for Research Institute according to published procedures (4) and used as antitumor studies. Animals were inoculated s.c. at the interscapular such in these studies. All other chemicals and reagents used were of region with 105 B16 melanoma cells on day 0 and treatment with DFMO analytical grade. was started on day 1. DFMO was administered p. o. at different concen Cells. B16-F, melanotic melanoma cells were kindly supplied by Dr. I. trations in aqueous solution as the sole drinking fluid. At the end of 21 J. Fidler, National Cancer Institute, Frederick Cancer Research Center, days the animals were sacrificed and tumors were dissected and Frederick, MD. The cells were cultured as monolayers at 37°Cin Eagle's weighed. A portion of the tumor was frozen at -70°C for polyamine and minimum essential medium supplemented with heat-inactivated calf tyrosi nase determinations. serum (10%), nonessential amino acids, sodium pyruvate, glutamine, and penicillin-streptomycin mixture in an atmosphere containing 5% CO2. Biochemical Assays In Vitro Cytotoxicity. Cytotoxicity of DFMO to B16 melanoma cells in culture was assessed by determining its effect on growth rate and cloning Tyrosinase Assay. The frozen cell and tumor tissues were homoge efficiency of the cells. Growth rate was estimated from periodic cell count nized in 4 volumes (w/v) of 25 mw Tris-HCI (pH 7.4) containing 0.5% data. Cell enumeration was done in a Coulter Counter after trypsinization Triton X-100. The homogenates were centrifugea at 25,000 x g for 30 of the cells. Cloning efficiency was evaluated according to the procedure min. The supernatant was used for the determination of tyrosinase described in detail earlier (27). activity according to the procedure of Pomerantz (28). Protein was determined by the method of Bradford (29). In Vivo Assays Polyamine Assay. The cell and tumor tissues were homogenized in Tumor. B16 melanoma (line f1) was propagated and maintained in 0.4 M perchloric acid and the supematants obtained after centrifugation vivo by serial transplantation of dissociated tumor cells into C57BL/6J were used for polyamine determination by dansylation and subsequent thin layer chromatography as described earlier (27). 8.0 RESULTS

"Ì6.0 The effect of DFMO (2.5 ITIM)treatment on the growth and cloning efficiency of B16 melanoma cells is presented in Chart o 1. The data indicate that DFMO inhibits the growth of melanoma f 4.0 cells (Chart 1/4).There was very little increase in the cell number in the DFMO treated cell population whereas an exponential O increase in cell number was observed in control cultures. To 2.0 determine further whether the inhibition of growth of the tumor cells was a cytostatic or cytotoxic effect of DFMO cloning efficiency of cells from control and DFMO treated population was determined. The results presented in Chart 1B indicate a time- IOO dependent Cytotoxicity of DFMO to B16 melanoma cells. At the end of 7 days DFMO treatment resulted in 90% cell killing as evidenced by the survival fraction of 10% compared to control cells. DFMO Cytotoxicity to B16 melanoma cells was accom panied by a rapid depletion of intracellular putrescine and sper- midine in the treated cells. A good correlation of the time

table 1 Effect of DFMOtreatmenton the polyamine levels otB16 melanomacells in culture Experimentaldetails are as described in the legendto Fig. 1. The values are an averageof two experiments. cells)Days1357TreatmentControl Polyaminelevels(mol/109

Days of DFMO Treatment DFMOControl 0.351.61 1.484.07 2.721.71 Chart 1. Effect of DFMO treatment on the growth and cloning efficiency of B16 melanomacells in culture. B16 Melanoma cells were plated at a density of 2.5 x 10scells/dish (100 mm) and treated with either regular mediumor medium contain DFMOControl 0.321.68 0.633.43 4.211.29 ing DFMO (2.5 mm).At the indicated times the cells were trypsinized and counted. For determining the cloningefficiency, cells were plated (400 cells/35 mm dish) and incubated at 37°Cin a 5% CO2 atmosphere. At the end of the incubation the DFMOControl0.131.33Not 0.295.51 1.393.25 colonies were fixed with formalin, stained with crystal violet, and counted under a dissecting microscope. The average number of colonies obtained in untreated control was taken as 100%. Routinely we obtain a plating efficiency of 50% values DFMOPutrescine2.83detectableSpermidine6.040.05Spermine2.030.49 which are means of two experiments. Bars, SE; O, control; •,DFMOtreated.

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4068 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. INHIBITION OF POLYAMINE BIOSYNTHESIS BY DFMO ON B16 MELANOMA

This inhibition also correlated well with a decrease in putrescine and spermidine concentration of the tumor tissue. Furthermore a 6-fold increase in the tyrosinase activity was observed in DFMO treated tumor compared to tumor obtained from control animals (Table 2).

DISCUSSION

The results of this study indicate that B16 melanoma is a sensitive tumor both in vitro and in vivo to polyamine depletion induced by DFMO treatment. DFMO was found to be cytotoxic to B16 melanoma similar to that of small cell lung carcinoma cells observed by Luk ef al. (5). DFMO treatment of B16 mela noma bearing mice also showed a dramatic reduction of 86% of tumor growth compared to control animals. B16 melanoma in mice seems to be one of the most sensitive tumors to DFMO treatment. Furthermore, treatment of melanoma cells both in vitro and in vivo caused a stimulation of tyrosinase activity and melanogenesis which was abolished upon supplementation of Days of DFMO Treatment putrescine both in vitro and in vivo, suggesting a critical role for Chart 2. Effect of DFMO treatment on the tyrosinase activity and melanin polyamines in the expression of these differentiation markers by content of B16 melanoma cells in culture. B16 melanoma cells were plated at a density of 2.5 x 10scells/100 mm dish. The dishes were divided in three groups. melanoma. Onegroup of dishes contained regular mediumand served as the control; a second Based on the data presented it is interesting to speculate that group was treated with DFMO (2.5 mu); the third group contained medium with the unique sensitivity of B16 melanoma to DFMO may be due to DFMO (2.5 mw) plus putrescine (50 tin). The cells were incubated at 37°Cina 5% a combination of antiproliferative effect and a concomitant induc COz atmosphere. At the indicated times the cells were trypsinized and counted and samples were frozen at -70°C for determination of tyrosinase activity and tion of differentiation. The fact that exogenous supplementation melanincontent. Tyrosine activity: O, control; •,DFMO;A, DFMO plus putrescine. of putrescine reverses the effects of DFMO supports this obser Melanincontent: D, control; •,DFMO;A, DFMO plus putrescine. vation. However, a similar expression of differentiated function by B16 melanoma cells has been reported following treatments dependent increase in the cytotoxicity of DFMO and decrease in with anthracycline antitumor agents (30). Recently Kapyaho ef the intracellular putrescine and spermidine of tumor cells was al. (31) have reported that methylglyoxal bis(guanylhydrazone) observed (Table 1). inhibited the growth of melanoma cells and induced melanin The results of the effect of DFMO on tyrosinase activity and accumulation without significantly reducing the polyamine levels. melanogenesis of B16 melanoma cells (Chart 2) indicate a 2- to These observations cannot rule out the possibility of induction 5-fold increase in the tyrosinase activity followed by 10-fold of tyrosinase and melanin accumulation as a result of antipro increase in the melanin content in DFMO treated cells compared liferative effect of the polyamine antimetabolites. It will be of to control cell populations. Furthermore, supplementation of interest to understand whether the expression of differentiated putrescine to DFMO treated cultures resulted in the reversal of phenotype is dependent on the antiproliferative effect of DFMO enzyme activity and melanin content to control values. or not. Our preliminary studies with B16 amelanotic melanoma, In order to determine whether the observations made in cell a tumor with negligible tyrosinase activity indicate that DFMO culture could be extrapolated in vivo B16 melanoma grown as a neither induces tyrosinase activity nor is cytotoxic as seen in its solid tumor in mice was treated with different doses of DFMO melanotic counterpart (unpublished data). The results of the (Table 2). A good dose response effect of DFMO was observed present study and that of Kaphyaho and Janne (26) indicate that both on the inhibition of tumor growth and polyamine levels polyamines play an important role in the growth and differentia (Table 2). A 29, 60, and 80% inhibition of tumor growth was tion of melanoma and also provide a good example of inhibition obtained at 0.5,1, and 2% DFMO in drinking water, respectively. of tumor growth by induction of cellular differentiation and it will

Table2 Effect of different doses of DFMOon the growth, polyamine levels, and tyrosinaseactivity ofB16 melanomain mice B16 melanomacells (1 x 105)were injected s.c. in the interscapular region of C57BL mice. The animals were divided into 4 groups. One group was put on normal drinking water which served as a control and the other 3 groups were allowed to drink water containing 0.5, 1, or 2% DFMO starting on Day 1. At the end of 21 days the animals were sacrificed and tumors were dissected and weighed. A portion of the tumor was used for polyaminedeterminations. concentration (mol/g) activity 5)Putrescine44.6(mean±SE;n = weight (g) (cpm x I0*/mg protein) TreatmentControl (mean8)6.70 ±SE;n = inhibition29 (mean4)118 ±SE;n = perniine337 ±61.0Not ±5.6 ±85.3 .4 ±71.7 DFMO (0.5%) 4.77 ±0.5" determined 9.87 + 1.7 155.6 ±22.6 679.5 ±49.2 DFMO(1%) 2.66 ±0.2* 60 Not determined 6.6 ±0.8 85.6 ±5.7 542.6 ±24.4 666 ±53.06Polyamine ±125"P

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4069 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. INHIBITION OF POLYAMINE BIOSYNTHESIS BY DFMO ON B16 MELANOMA be of interest to see whether human malignant melanoma would 14. Gaziti, Y., and Friend, C. Polyamine biosynthesis enzymes in the induction and inhibition of differentiation in Friend erythroleukemiacells. Cancer Res., be sensitive to DFMO treatment. 40:1717-1732,1980. 15. Huberman,E., Weeks, C., Heirmann,A., Callihan,M., and Slaga,T. Alterations in polyaminelevelsinduced by phorbol diesters and other agents that promote ACKNOWLEDGMENTS differentiation in human promyelocytic leukemia cells. Proc. Nati. Acad. Sci., USA, 78: 1062-1066,1981. The authors would like to thank A. Rosenbergerand G. Davisfor their technical 16. Luk, G. D., Civin, C. L., Weissman, R. M., and Baylin, S. B. Omilhine help and L. Parro«andJ. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. Effect of Inhibition of Polyamine Biosynthesis by dl-α -Difluoromethylornithine on the Growth and Melanogenesis of B16 Melanoma in Vitro and in Vivo

Prasad S. Sunkara, C. C. Chang, N. J. Prakash, et al.

Cancer Res 1985;45:4067-4070.

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