Inhibition of EMT6 tumor growth by interference with polyamine biosynthesis ; Effects of α-difluoromethyl- ornithine, an irreversible inhibitor or ornithine decarboxylase

Pergamon Press

Life Sciences, Vol . 26, pp . 181-194 Printed in the U .S .A .

INHIBITION OF EMT6 TUMOR GROWTH BY INTERFERENCE WITH POLYAMINE BIOSYNTHESIS ; EFFECTS OF a-DIFLUOROMETHYLORNITHINE, AN IRREVERSIBLE INHIBITOR OF ORNITHINE DECARBO%YLASE . Nellikunja J . Prakanh, Paul J. Schechter l , Pierre S . Manvont, Jeffrey Grove, Jan Roch-Wener and Albert Sjoerdsma Centre de Recherche Merrell International, 16, rue d'Ankara 67084 Strasbourg Cedex, France (Received in final form November 28, 1979)

Summary a-Difluoromethylornithine (a-DFMO), an enzyme-activated irreversible inhibitor of omithine decarboxylase (ODC), retarded the growth rate of EMT6, a murine mammary sarcoma, in tissue culture . When female BALB/C mice were inoculated subcutaneously with EMT6 cells, administration of a-DFMO as a 3 % solution'in the drinking water beginning 5 days after tumor inoculation resulted in an 80 Z inhibition of tumor weight gain by day 27 compared to controls . Thin treatment regimen, equivalent to 4 .4 g a-DFMO/kg/day, decreased tumor ODC activity, stimulated S-adenosyl-L-methionine decarboxylase (SAM-DC) activity and markedly decreased tumoral putreacine and spermídine, but not epermine, concentrations . The tumor growth inhibitory effects of a-DFMO were similar to those obtained with 4 weekly doses of cyclophoephamide (100 mg/kg i .p . beginning on day 6 poet-inoculation) . The combination of cyclophosphamide plus a-DFMO caused the same or greater inhibition of tumor growth than either treatment alone . When the SAM-DC and diamine oxidase inhibitor, 1,l'-((methylethanediylidene)-dinitrílo) bin (3-aminoguanidine), was added to a-DFMO treatment, tumor SAM-DC activity, putreacine and epermidine concentrations, but not ODC activity, returned to control values and the anti-proliferatioe effects of a-DFMO were reversed . These results suggest that a-DFMO treatment is as effective non-toxic method of inhibiting tumor growth by a mec~tanism involving polyamine depletion. The association of an increased rate of polyamine biosynthesis and of accumulation of putreacine and apermidine with rapid mammalian cell grwth and proliferation has frequently been observed (1,2) . One of the earliest events occurring upon transformation of cells is an increased activity of omithine decarboxylase (ODC ; E .C . 4 .1 .1 .17) followed by an accumulation of polyamines (3,4) . Hence, much interent has centered on polyamine metabolism is neoplaaia. 1 . To whom requests for reprints should be addressed .

0024-3205/80/030181-14$02 .00/0 Copyright (c) 1980 Pergamon Presn Ltd

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Ne have previously demonstrated that interference with polyamine biosynthesis by a-difluoromethylornithine (a-DFMO, RMI 71 .782), a specific and nontoxic enzyme-activated irreversible inhibitor of ODC, prolonged survival in L1210 leukemia-bearing mice (5) . In the present investigation, we found an inhibitory action of a-DFMO on the proliferation of EMT6 cells, a murine sarcoma (6,7), grown in cell culture . The effect of a-DFMO, alone and in combination with cyclophosphamide, on the growth of EMT6 tumor in mice and on tumor polyamine metabolism was then established . Finally because of an observed stimulatory effect of a-DFMO treatment on the activity in tumor of a second enzyme involved in polyamine biosynthesis, namely S-adenosyl-L-methionine decarboxylase (SAM-DC ; E .C . 4 .1 .1 .50), an inhibitor of this enzyme, 1,1(methylethane-diylidene)-dinitrilo) bis (3-aminoguanidine) (MBAG), was also studied ín a-DFMO-treated animals . Materials and Methods Materiale 14 ~ DL-~l-C ornithine (specific activity, 59 mCi/mmol) and S-adenosyl-L~1-C1 4 ~ -methionine (specific activity, 58 mCi/mmol) were purchased from the Radiochemical Centre (Amersham, England) . a-DFMO and MBAG were synthesized in our Centre by published procedures (8,9) . Cyclophosphamide was purchased from Laboratoire Lucien (Colombes, France) and S-adenosyl-L~methionine from Sigma Chemical Co . (St . Louis, MO ., U .S .A .) . All other chemicals were of the highest purity available from E . Merck (Darmstadt, Germany) . Cell Culture Mouse mammary EMT6 cells (donated by Dr . E . Frindel, Institut de Radiobiologie Clinique, Villejuif, France) were grown as described (7) in Weymouth's medium supplemented with 15 % fetal calf serum (Gibco, Grand Island, N .Y .) . For in vivo transplantation, cultures at near confluency were trypsinized and suspended in phosphate-buffered saline to a density of 106 cells/ml prior to inoculation . In vitro cell~roliferation EMT6 cells growing in monolager were trypainized, counted and 1 .12 R 10 5 cells were seeded in duplicate 80 mm dishes . 30 min later 5 mM a-DFMO was added . Cell counts were determined at the indicated times after trypsinization . Cell viability was estimated by the trypan blue exclusion méthod . Detached cells, which represented less than 5 z of the total cell count both in treated and untreated cultures, were neglected . Animals Female BALB/C mice, initial body weight 18-22 g, were obtained from Charles River (France) . They were housed in groups of 20 in plastic cages with sawdust bedding with free access to food . Drinking fluid was available ad libitum from plastic water-bottles . Humidity (45-55 %), temperature (21-23 ° ) and 12 hr light cycle (beginning at 6 a .m .) were maintained constant throughout the investigation . In vivo tumor proliferation Animals were inoculated subcutaneously in the interscapular region with 105 cells/mouse . Animals were sacrificed by cervical dislocation or exsanguination by cardiac puncture under ether anaesthesia . Tumor growth was monitored by weighing the tumor samples . Immediately after the removal of the tumors,

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3-4 mm midline slices were cut, frozen in liquid nitrogen and stored at - 70°C until analyzed for polyamine content and enzyme activities . Enzyme activities were determined within 24 hours of tumor sampling . Administration of drugs Cyclophosphamide was injected i .p . as an aqueous solution in a volume of 10 ml/kg . MBAG was administered similarly as a suspension ïn water . a-DFMO was administered orally by offering a 2 or 3 Z aqueous solution of the compound as the sole drinking fluid . Daily fluid intake/mouse was estimated by measuring the total fluid intake per cage and dividing by the number of mice contained therein . Biochemical measurements Tumors were thawed and homogenized in 9 volumes of ice-cold 0 .1 M phosphate buffer, pH 7 .2, containing 1 mM dithiothreitol, 0 .1 mM Na2 EDTA and 10 uM pyridoxal phosphate in a glass homogenizer fitted with a Teflon pestle . A por tion of the homogenate was mixed immediately with an equal volume of 0 .4 M HC104 and centrifuged . The supernatant from this perchloric acid extract was analyzed for polyaminea and a-DFMO by published procedures (5,10) . The remainder of the homogenate in phosphate buffer was centrifuged at 40,000 % g at 0°C for 1 hour . Aliquots of the supernatant were taken for determination of ODC and SAM-DC activities by the methods described in an earlier report (5) . Values for enzyme activities are expressed as nmol/g/hr . Hematological measurements Erythrocyte and leukocyte counts of heparinized blood samples, obtained from animals exsanguinated by cardiac puncture under ether anesthesia, were determined employing a model ZB1 Counter Counter (Coultronics, France) . Hemat ocrits were estimated by centrifugation of whole blood samples and subsequent measurement of the packed cell volume . Statistical analysis Significant differences of group means were assessed using Students t test (2-tailed) or one~ay analysis of variance as appropriate . Results Experiment I : Effect of a-DFMO on EMT6 cells in culture EMT6 cells grown in monolayer cultures have a lag phase of growth of about 24 hours (Fig . 1) followed by a rapid exponential phase with a population doubling time of about 11-12 hours . When a-DFMO to produce a final concentration of 5 mM is added to the culture 30 minutes after seeding, the lag phase is not modified, but a striking and progressive decrease of the growth rate is observed thereafter . a-DFMO affected neither cell attachment properties nor cell viability . Experiment II

: Effect of a-DFMO on EMT6 tumor in mice

Mice were inoculated subcutaneously with EMT6 tumor cells (105 cells/mouse) and were given a-DFMO as a 3 % solution in the drinking water beginning 5 days following inoculation . The volume of fluid consumed by mice offered 3 x a-~FMO (2 .92 ± 0 .14 ml/mouse/day ; mean ± SEM) was less than that consumed by control mice drinking tap water (3 .34 ± 0 .09 ml/mouse/day) . For a 20 g mouse this cor-

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FIG . 1 Inhibitory effect of 5 mM a-DFMO on EMT6 cell proliferation in vitro . Values represent the average of two dewations . Duplicates did not differ by more than f0 % from each other . responds to a dose of 4 .38 ± 0 .21 g/kg/day of a-DFMO . a-DFMO treatment dramatically retarded tumor growth (Fig . 2) . On day 27 the average tumor weight of control mice was approximately S times that of treated animals . Tumor weight of a-DFMO-treated mice did not change significantly between days 18 and 32 (F 20 ~ 1 .225), whereas control tumor weights more than doubled during this interval .

3

Tumor ODC activity increased in control animals to a peak at day 7 (Fig . 2) . Enzyme-activity then decreased by day 12 to a stable low level . This peak of ODC activity was abolished by a-DFMO treatment . In tumors of treated mice ODC activity was decreased at day 7 and remained lower than that in control animals . On the other hand, SAM-DC activity of control tumors remained low throughout the experiment while in a-DFMO-treated animals tumoral SAM-DC activity increased to a maximum by day 12 and remained elevated .

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FIG. 2 Inhibition of EMT6 tumor in BALB/C mice (105 cella subcutaneously/mouse) by a-DFMO (3 % in drinking water) and effect of a-DFMO treatment on tumoral ODC and SAM-DC activitîea . Polyamine concentrations in control tumors were relatively stable throughout the experiment (Fig . 3) . Putreacine and epermidine concentrations in tumor tissue of a-DFMO-treated mice were 3 to 40-fold lower than those of controls . Sperraine concentrations showed no difference between the two groups . The concentrations of a-DFMO in the tumors of treated mice varied between 135 nmoles/g on day 12 to 328 nmolea/g on day 18 with an average of 265 nmoles/g for all 6 time points sampled .

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Experiment III : Effect of a~FMO and cyclophoaphamide on EMT6 tumor in mice Mice, inoculated subcutaneously with 10 5 EMT6 cells/mouse, were divided into four groups . One group (controls) received tap water ; one group 3 X a-DFMO as the sole source of drinking fluid beginning day 6 after inoculation one group was given tap water and injected with cyclophoaphamide, 100 mg/kg i .p . once weekly for 4 weeks ; and the final group received both a-DFMO and cyclophoaphamide . The average daily fluid intake of all four groups is given in Table I . Mice given 3 % a-DFMO consumed less fluid than control animals . Mice treated with cyclophosphamide drank similar quantities as control mice . Cyclophosphamide-treated animals usually demonstrated a slight decrease in fluid intake for the 2 days following each i .p . dose . Mice given both a-DFMO and cyclophosphamide had a mean daily fluid intake less than the other groups . With these animals the decrease observed for 2 days following each cyclophosphamide dose was exaggerated .

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TABLE I Daily Fluid and a-DFMO Intake (mean ± S .E . ; n = 20-28) of Mice given 3 % a-DFMO in Drinking Water with and without Concomitant Cyclophosphamide Therapy.

Treatment

Daily Fluid Intake (ml/mouse)

Daily a-DFMO Intake (g/kgt)

Control (water)

3 .47 ± 0 .10

-

a-DFMO

2 .79 ± 0 .09x

4 .19 ± 0 .14

Cyclophosphamide (100 mg/kg i .p . X 4 doaea)

3 .40 ± 0 .09

a-DFMO + Cyclophoaphamide

1 .95 ± 0 .10-~

t ""

2 .93 ± 0 .15

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FIG . 4 Inhibitory effect of a-DFMO (3 X in drinking water) and cyclophosphamide (100 mg/kg i .p . R 4 doses - arrows) alone and in combination, on EMT6 tumor growth in mice (10~ cella subcutaneously/mouse) . Values represent means ± S .E . (n a 6) .

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EMT6 tumor growth in control animals and in animals given 3 % a-DFMO was similar to that observed in Experiment II, demonstrating the reproducibility of this tumor model and of the effects of a-DFMO (Fig . 4) . Between days 18 and 35 tumor size in a-DFMO-treated mice was stationary (F4 30 = 0 .790) and by day 35 the average weight was approximately 1/4 of control tumors . Tumor growth in mice treated with 4 doses of cyclophoaphamide, 100 mg/kg i .p ., was parallel to that of a-DFMO-treated animals . This dose and schedule of cyclophosphamide were found in pilot experiments to be the maximum possible, since greater or more frequent doses invariably caused mortality in tumor-bearing animals . Mice treated with both a-DFMO and cyclophoaphamide had tumors weighing slightly less than those treated with a-DFMO alone . This difference was statistically significant (p < 0 .02) from day 28 . TABLE II ODC Activity (nmoles/g/h) in EMT6 Tumore (10 5 cells aubcutaneously/SALB /C mouse) after Treatment with a-DFMO and Cyclophoephamide .

Days

Treatment

PostInoculation

a-DFMO~" Control

a-DFMO~~

Cyclophosphamide t

+

Cyclophoaphamide t

5

10 .86 ± 1 .26

-

7

25 .75 ± 4 .02

-

12

9 .58 ± 2 .35

3 .33 ± 0 .37

21 .61 ± 2 .67

4 .21 ± 0 .31

18

3 .69 ± 0 .53

1 .44 ± 0 .20

6 .84 ± 1 .10

2 .10 ± 0 .20

25

4 .99 ± 1 .14

1 .09 ± 0 .17

3 .14 ± 0 .70

0 .88 ± 0 .19

28

2 .46 ± 0 .53

0 .89 ± 0 .16

0 .65 ± 0 .08

1 .13 ± 0 .20

35

4 .09 ± 1 .45

1 .13 ± 0 .22

2 .08 ± 0 .49

0 .49 ± 0 .11

Values are means - S .E . (n = 6) 3 % in the drinking water beginning on day 6 t 100 mg/kg i .p . on days 6, 14, 21 and 28 ODC activity in tumors of control mice, as in the earlier experiment, increased within the first several days following inoculation and reached a peak by day 7 (Table II) . Thereafter, enzyme activity began to decrease . T~ora of a-DFMO-treated mice had a lower ODC activity than those of control mice . In tumors of cyclophosphamide-treated mice, the elevation in ODC activity was apparently prolonged, possibly reflecting rapid re~rowth of the surviving fraction of cells following chemotherapy . This prolongation of the period of high ODC activity induced by cyclophosphamide was abolished in tumors of mice treated with a-DFMO plus cyclophosphamide, suggesting that regrowth of the surviving fraction was retarded by the a-DFMO . a-DFMO treatment increased SAM-DC activity of the tumor (Table III), where-

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se cyclophosphamide exerted little influence . The combination of a-DFMO and cyclophoaphamide produced lean of an increase in tumor SAM-DC activity than a-DFMO alone . Thin may reflect the lower dose of a-DPMO cons~ed by these animals (Table I) or may be attributable to effects of the cyclophoaphamide . TABLE III SAM-DC Activity (nmoles/g/h) in EMT6 Tumore (105 cells aubcutaneously/BALB/C mouse) after Treatment with a-DPMO and Cyclophosphamide .

Treatment

Days PostInoculation

a-DFMOx Control

a-DFMO~"

Cyclophosphamidet

+

Cyclophoaphamide i

5

299 .6 ± 22 .5

7

159 .9 ± 14 .0

12

126 .7 ± 20 .1

1728 .2 ± 201 .8

230 .5 ± 21 .8

18

127 .3 ± 28 .5

1326 .2 ± 131 .9

169 .3 ±

5 .9

1063 .5 ± 83 .9

25

185 .0 ± 20 .2

1084 .7 ± 117 .7

220 .5 ± 18 .8

485 .4 ± 40 .5

28 35

57 .0 ±

8 .8

898 .6 ± 124 .8

208 .7 ± 30 .8

1312 .1 ± 108 .7

845 .0 ± 96 .6

3 .0

463 .3 ± 71 .9

185 .3 ± 12 .2

497 .6 ± 66 .8

35 .2 ±

Values are means ± S .E . (n a 6) 3 I in the drinking water beginning on day 6 t 100 mg/kg i .p . on days 6, 14, 21 and 28 Tumor polyamine concentrations of control and a-DFMO-treated mice (Pig . 5) were similar to those found in Experiment II . a~FMO decreased putreacine and epermidine but not spermine concentrations . The early tumors from cyclophos phamide-treated mice had higher concentrations of putreacine and spermidine than controls, probably due to drug-induced cell lose and subsequent regrowth of the surviving fraction (11) . At later times, tumors from cyclophosphamide-treated mice had lower concentrations of putreacine and spermidine than those of controls . Mice treated with both a-DFMO and cyclophosphamide had tumor putreacine and spermidine concentrations similar to or slightly greater than a-DFMOtreated animals . Sperraine concentrations within each group changed little throughout the course of the experiment . No significant differences in hematocrit or in erythrocyte counts were apparent for any group of animals at the time of sacrifice, i .e ., days 0, 7, 12, 18, 25, 28 and 35 post-inoculation . All tumor-bearing animals manifested a leucocytosis beginning on day 25 or 28 post inoculation. Leucocytosis was greatest in those animals treated wíth cyclophoaphamide (up to 130,000 cells/ mm3) and consisted primarily of hypersegmented polymorphonucleocytea .

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FIG . 5 Effect of a-DFMO (3 % in drinking water) and cyclophoaphamide (100 mg/kg i .p . R 4 doses = arrows), alone and in combination, on polyamine concentrations of EMT6 tumors in mice . Values represent means ± S .E . (n ~ 6) . Experiment IV : Effect of a-DFMO and MBAG on EMT6 tumor in mice In an attempt to suppress the increase in tumoral SAM-DC activity found following a-DFMO administration, a-DFMO treatment was combined with MBAG, an irreversible SAM-DC inhibitor (12) . In a preliminary experiment, it was found that mice treated äaily with MBAG did not readily accept 3 % a-DFMO in the drinking water . Therefore, in this study a 2 % solution of a-DFMO was used . Mice, inoculated as before, were divided into three groups and were allowed either tap water (controls) ; 2 X a-DFMO as the sole source of fluid beginning

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a-Difluoromethylornithine and Tumor Growth

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on day 6 post-inoculation ; or 2 % a-DFMO plus MBAG 25 mg/kg/day i .p ., beginning on day 6 . In this experiment control mice drank an average of 3 .4 ml/day/ mouse of water, animals given 2 % a-DFMO drank 3 .3 ml/day/mouse (equivalent to a dose of 3 .3 g a-DFMO/kg/day for a 20 g mouse) and animals treated with MBAG drank 2 .8 ml/day/mouse (or 2 .8 g a-DFMO/kg/day) . 2 Z a-DFMO beginning on day 6 post-inoculation produced a decrease of 43 % in tumor weight by day 14 relative to control mice (Table IV) . Tumor ODC activity was leas than 1/3 that of control mice, SAM-DC activity was markedly stimulated and putrescine and epermidine concentrations were decreased . Unlike in experiments II and III where 3 Z a-DFMO was used, in this study tumor spermine concentrations were significantly increased . The addition of MBAG to a-DFMO treatment did not alter ODC inhibition but, as expected, blocked the SAM-DC increase induced by a-DFMO . In addition, however, putrescine and spermidine concentrations were restored to control values . This unexpected effect on .polyamine concentrations probably reflects the inhibitory activity of MBAG on diamine oxidase (13), the enzyme involved in polyamine catabolism . Associated with these biochemical events, there was an antagonism of the effect of a-DFMO on tumor growth . Diacusaion Continuous polyamine biosynthesis appears to be required to maintain maximum rates of cell proliferation . Aa increased polyamine content in tumorbearing tíasues of both animals and humane has been noted (14-16) and corre lations have been made between tumor growth rate and polyamine content (2,17) . Although known effective anticancer agents influence tumor polyamine metabolism (18), few attempts have been made to influence tumor growth therapeutically by specific inhibition of polyamine biosynthesis . DL-~t-hydrazino-d-aminovaleric acid, a competitive inhibitor of ODC, decreased the growth of sarcoma-180 in mice (19) . Methylglyoxal-b is (guanylhydrazone) or MGBG, a well-known competitive inhibitor of SAM-DC, prolonged survival of mice inoculated with L1210 leukemia (18,20), sarcoma-180 (21), and other leukemias (21) and has been used clinically to treat acute myelocytic leukemia, malignant lymphoma and myeloma (22) . Clinical application of this agent was hampered, however, by frequent aad severe toxicity (23) . a-DFMO, as reported previously, is an irreversible enzyme-activated inhibitor of ODC in vitro (8) . When administered ey~temically to rata, it inhibits tissue ODC actiey, and decreases polyamine concentrations (24) . In tissue culture, a-DFMO decreases proliferation in a variety of cell lines (25,26) . In addition, a-DFMO treatment prolongs survival of mice bearing L1210 leukemia (5) . For the present study, the EMT6 tumor was chosen . This mammary sarcoma is an example of a transplantable in vivo - in vitro cell line advocated as a model system for experimental oncology (27 . We first demonstrated that a~FMO produces a decrease in the rate of EMT6 growth in cells in culture . These results are similar to the anti-proliferatioe effects of the same concentration of a-DFMO on cultures of rat hepatoma culture (HTC) cells, L1210 leukemia cells, MA 160 prostate adenoma cells and human fibroblasts previously reported (25,26) . When injected subcutaneously into female BALB/C mice, EMT6 cells (105 cells/ mouse) produce a palpable tumor within 5-6 days . If a-DFMO treatment is begun at this time, the subsequent growth of the EMT6 tumor is markedly retarded . Tumor growth retardation is associated with decreased ODC activity, increased SAM-DC activity and decreased tumor concentrations of putrescine and apermídine . Qualitatively similar biochemical effects following a-DFAiO have been observed

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Vol . 26, No . 3, 1980

Vol . 26, No . 3, 1980

a-Difluoromethylomithine and Tumor Growth

19 3

in vitro (26) and in vivo .(5) . A causal relationship between depletion of tissue polyamine concentrations and decreased proliferation following a-DFMO would be suggested if repletion of polyamines would reverse the anti-proliferative effects . Such reversal has been demonstrated in vitro . The additîon of .putrescine, spermidine or spermine to,HTC cells whose growth had been inhibited with a-DFMO, restored maximum rates of cell proliferation (28) . Analogous repletion experiments are not possible in vivo due to the inherent toxicity of systematically administered polyaminea the intact animal (29) . Using the SAM-DC and diamine oxidase inhibitor, MBAG, we have been able to prevent the decrease in tumor putrescine and spermidine concentrations produced by a-DFMO . The maintenance of the polyamine concentrations at control levels was accompanied by an antagonism of the anti-proliferative effects of a-DFMO on tumor growth . It has been suggested that ODC activity itself and not necessarily polyamine biosynthesis may be directly related to RNA synthesis and, by extension, to cell growth (30,31) . This does not appear to be the case in the present studies since MBAG treatment restored tumor growth without influencing the a-DFMOinhibited ODC activity . Because of the dual effect of MBAG on SAM-DC and diamine oxidase, it would be of interest to repeat these studies using a-DFMO plus specific inhibitors of diamine oxidase .

in

It has previously been shown (11) that cyclophoephamide, 100 mg/kg, injected i .p . 5 days after EMT6 tumor inoculation, produces a growth delay of approximately 6 days when Manor volume is used as a parameter . Hence, in the present study weekly cyclophoephamide treatment in tumor bearing mice was compared to a-DFMO treatment . The inhibitory effects of a-DFMO on tumor growth were equivalent to the effects of the maximally tolerated cyclophoephamide regime . When a-DFMO and cyclophoephamide treatments were combined, tumor growth inhibition was equal or greater than either treatment alone . a-DFMO appears to decrease cell replication without cytotoxicity . Preliminary in vitro experiments using time-lapse photomicrographic techniques indicate that a-DFMO markedly and progressively increases EMT6 intermitotic times (10-fold after the fourth generation) with no evidence of cell toxicity (M .Collyn d'Hooghe and P .S . Manvont, personal communication) . This would explain the apparent lack of additive effects on cell growth retardation when a-DFMO treatment is combined with that of a cyclerspecific agent like cyclophoephamide . It is possible that further alterations of doses and treatment schedules of a-DFMO plus cytotoxic agents will demonstrate more profound synergistic anti-tumor effects . Our results suggest that a-DFMO treatment nearly arrests EMT6 proliferation in vivo . Hence, a greater cell kill might be expected if a-DFMO treatment was - d~econtinued for an interval sufficient for the cells to enter a proliferative phase prior to cycle-specific cytotoxic therapy . The absence of cytotoxicity with a-DFMO is probably responsible for its extremely low level of toxicity in animals . The acute LDgp in rats and mice exceeds 3000 mg/kg i .p . (5) and 5000 nv8/kg orally . Non-tumor bearing mice given 3 x a-DFMO in drinking water for over 4 weeks displayed a weight gain similar to water-drinking controls with no evidence of toxic effects . The growth retarding effects of a-DFMO on tumors and its relative lack of toxicity may make this inhibitor of ODC a useful adjuvant to current cancer chemotherapeutic regimens which generally utilize cytotoxic agents . Additional studies will be necessary to establish the optimal combinations and treatment sequences of therapeutic possibilities .

194

a-Difluoromethylornithine and Tumor Growth

Vol . 26, No . 3, 1980

Acknowledgments The skillful technical assistance of Madame N . Nagy and the secretarial help of Mademoiselle C . Sche penberger are gratefully acknowledged . a-DFMO and MBAG were synthesised by Dr . P . Bey . References l . 2. 3. 4. 5. 6. 7. 8, 9, 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 .

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