Involvement of Histamine in Growth of Mouse and Rat Tumors

[CANCER RESEARCH 44, 639-645, February 1984]

Involvement of Histamine in Growth of Mouse and Rat Tumors: Antitumoral Properties of Monofluoromethylhistidine, an Enzyme-activated Irreversible Inhibitor of Histidine Decarboxylase

Jacques Bartholeyns1 and M. Bouclier

Centre de Recherche Merrell International, 16, rue d'Ankara, 67084 Strasbourg-Cedex, France

ABSTRACT proliferating tissues [e.g., rodent fetal tissues, tissues involved in wound healing, skin under the influence of tumor promoters, The present study suggests that newly synthesized histamine sarcomas, and Walker carcinosarcoma (9, 14, 17, 34, 35)]. It is involved in the development of some animal tumors (e.g., has been suggested that newly synthesized (nascent) histamine Lewis lung carcinoma in mice and Morris hepatoma in rats). A could be involved in the proliferation of tumors or tissues marked induction of histidine decarboxylase (HOC) and an in undergoing rapid cell division (11, 19). In support of this, recent crease in the histamine concentration were observed in the data have indicated that anticancer effect can be achieved with tumors approximately 1 week after inoculation, and there were cimetidine, a selective H2 receptor antagonist, in mice bearing parallel increases in ornithine decarboxylase activity and the carcinoma, lymphoma, or fibrosarcoma (13, 23). concentrations of polyamines. The H2 receptor antagonist, ci- It is now generally established that rapid tissue growth is metidine, significantly reduced tumor growth in the animal models associated with elevated activity of ODC (EC 4.1.1.17) and, as while the H-, receptor antagonist, dexchlorpheniramine, had no a result, with increased production of polyamines (18, 21, 24, effect, suggesting that histamine could act via H2 receptor sites. 25, 28). We have therefore compared the induction of ODC and Extensive depletion of tumor histamine induced by local injection HOC and the local increase in polyamines and histamine levels of Compound 48/80 did not result in a significant cytostatic after inoculation of tumors in 2 different animal tumor models. effect. The antitumoral effect of antihistamine drugs was investigated Monofluoromethylhistidine (MFMH), an enzyme-activated irre to clarify the possible role of H, and H2 receptors. The effect of versible inhibitor of HOC, retarded the growth of hepatoma tissue local histamine release and depletion by Compound 48/80 was culture cells grown in culture, and when infused s.c. at 60 mg/ studied to differentiate the possible role of nascent histamine kg/day it greatly inhibited the development of tumors induced from that of the histamine stored within mast cells. i.m. by hepatoma tissue culture cells in Buffalo rats. MFMH also Finally, the development in our laboratories of MFMH (MDL had pronounced antitumoral effects on EMT6 sarcomas and 72209), an enzyme-activated irreversible inhibitor of HOC, Lewis lung carcinomas in mice, which were associated with prompted us to examine its effects on animal tumor models and inhibition of HOC and depletion of the histamine content of the to attempt to correlate these effects with selected biochemical tumors. alterations. The effects observed when MFMH was used in These cytostatic effects were clearly enhanced when MFMH combination with the irreversible inhibitor of ODC, DFMO (MDL was combined in therapy with the specific ornithine decarboxyl 71782), were also investigated in order to explore the effects on ase inhibitor, DL-a-difluoromethylornithine. The antitumoral ef tumor growth of impairment of the de novo biosynthesis of both fects of the combination were associated with marked decreases histamine and the polyamines. in the tumor histamine and putrescine contents. It is proposed that nascent histamine, like newly synthesized putrescine and MATERIALS AND METHODS spermidine, plays a role in the rapid proliferation of animal tumors. Inhibition of HOC by essentially nontoxic drugs such as MFMH Animals. Female C57BL mice (18 to 20 g) from Charles River were could represent a novel approach to the control of neoplastic used for the LL carcinoma. Male Buffalo rats (initial body weight, 160 to growth. 200 g) bred in our center were used for the experiments on solid tumors induced by HTC cells. The animals were housed in metal cages with free access to food and water. Fluid intake and body weight were measured INTRODUCTION at regular intervals. Room temperature (21-23Â°), humidity (45 to 55%), and a 12-hr light cycle (beginning at 6 a.m.) were kept constant through The biogenic amine, histamine, is known to be involved in out the investigations. inflammation, allergic reactions, and gastric acid secretion (11). Cells and Tumors. HTC cells, derived from Morris hepatoma 7288C Occasional indications can be found in the literature of high induced in Buffalo rats, were grown in spinner culture (16). Cells were histamine levels in some tumors [e.g., mice bearing C3H fibro harvested from their culture medium, counted, and washed twice with sarcoma or 3-methylcholanthrene-induced sarcoma and rats with 0.9% NaCI solution prior to use. The tumors were induced in Buffalo aflatoxin-induced hepatoma (7) or adenocarcinoma of the thyroid rats by i.m. injection into the leg of 2 x 106 HTC cells in 0.2 ml of 0.15 (22)]. The enzyme which catalyzes the formation of histamine M NaCI solution (2). Animals were examined every week for tumor from histidine, HOC2 (EC 4.1.1.22), is also high in several rapidly growth, and the diameter of their legs at the tumor level was measured with calipers in 2 perpendicular directions (breadth and width). The Received May 24, 1983; accepted October 5, 1983. tumoral cross section was considered an ellipse, and the following ' To whom requests for reprints should be addressed. 2The abbreviations used are: HOC, histidine decarboxylase; ODC, ornithine nithine; LL carcinoma, Lewis lung carcinoma; HTC cells, hepatoma tissue culture decarboxylase; MFMH, monofluoromethylhistidine; DFMO, DL-a-difluoromethylor- cells; SAMDC, S-adenosyl-L-methionine decarboxylase.

FEBRUARY 1984 639

formula was used to calculate its size: England. MFMH (MDL 72209) and DFMO (MDL 71782) were synthesized in this center. 7r/4.(i,f2-C,C2) RESULTS where f, and f2 are the perpendicular axes of the tumoral left leg and c-, and c2 are the perpendicular axes of the control right leg. De Novo Synthesis of Putrescine and Histamine in Buffalo EMT6 sarcoma cells were grown in culture as described previously Rats with HTC Tumors and C57BL Mice with LL Carcinoma (25). BALB/c mice were given injections of 105 cells/mouse in the interscapular region. The mice were sacrificed by cervical dislocation, Buffalo rats were given i.m. injections of HTC cells in the leg. and the tumors were removed and weighed (2). The tumor size increased more or less linearly with time starting LL carcinoma (3LL tumors, LL57B004-005) was propagated and 5 days after inoculation. HOC and ODC activities, which were maintained in vivo s.c. or i.m. in C57BL mice (1). The tumors were cut very low in the muscle, were markedly increased in the tumor a into small pieces, homogenized in 10 volumes of 0.9% NaCI solution by few days after inoculation, reaching a maximal value around Day 2 strokes of a Potter-Elvehjem homogenizer at 500 rpm, and filtered on 8 before decreasing again (Chart 1). The time of induction of the gauze. Tumors were induced in mice by i.m. injection into the leg of 5 x 105 cells in 0.05 ml 0.15 M NaCI. The tumor could be detected from Day 2 decarboxylases corresponded to the appearance of a macro scopic tumor in the muscle. The increase in putrescine content 8 and was measured with calipers using the technique described above followed closely the induction of ODC (Chart 1). Similarly, there for HTC tumors. The animals were sacrificed after 20 days, and their lungs were examined under a magnifying lens for the presence of was an early increase in tumor histamine concentration which mÃ©tastases. corresponded to the period of induction of HOC. There was also Biochemistry. Tissues were homogenized in 9 volumes of ice-cold an increase in histamine concentration later in tumor develop 0.1 M phosphate buffer (pH 7.2) containing 1 mw dithiothreitol, 0.1 mw ment, between 18 and 28 days after inoculation, which was not disodium EDTA, and 10 /JM pyridoxal phosphate and centrifuged. The associated with an increase in HOC (Chart 1). ODC and SAMDC activities of the supernatants were measured accord C57BL mice were given i.m. injections of LL carcinoma cells. ing to published methods (24, 25). Polyamines were analyzed by pub A discrete tumor which grew rapidly thereafter could be detected lished procedures (10) on supernatants from tumor homogenates follow at Day 8. Twenty days after inoculation, the lung contained 14 ing protein precipitation with 0.4 M perchloric acid. DNA was measured Â±2 (S.E.) mÃ©tastases.The very low ODC activity in the muscle according to the method of Burton (8), and protein was measured contrasted markedly with that of the tumor part. The tumor ODC according to the method of Lowry ef al. (20). Histamine content was measured on the supernatant obtained after activity increased rapidly one week after inoculation, reached a centrifugation of the crude tumor homogenates by a modification of an enzymatic isotopie assay (32). Supernatant (20 ^l) was incubated in a tube containing 30 n\ of the following reagent mixture: 10 Â¿igofhistamine W-methyl transferase prepared as described previously (24); 0.5 nC\ of 5OO I- 125 S-adenosyl-L-[mef/7y/-3H]methionine (final concentration, 2.5 x 10~5 M); o and 0.1 M phosphate buffer (pH 7.9). After 1 hr incubation at 37Â°,100 n\ 400 100 m of 1 N NaOH saturated with NaCI were added, and the methyl histamine formed was extracted into 1 ml of chloroform. The chloroform layer was 300 75 Â» washed with 100 n\ 0.1 N NaOH saturated with NaCI and then was evaporated in an air current. The residue was dissolved in a scintillation cocktail, and the radioactivity was counted (31). HOC activity was 200 determined on the crude tumor homogenate, using a modification of a â€¢ highly sensitive radiochromatographic procedure (3). The incubation 100 25 > mixture (70 M!)consisted of 50 Â¡Aoftumor homogenate, 1 ^Ci of purified L-[2,5-3H]histidine (about 2 x 10"11 mol), 10~5 M pyridoxal phosphate, 10~4 M histamine, and 5 x 10~2 M of phosphate buffer (pH 7). After incubation for 1 hr at 37Â°, the enzymatic reaction was stopped by addition of 10 p.\ of 2.4 M perchloric acid. After addition of 600 n\ of 0.2 M Tris-HCI buffer (pH 8), the [3H]histamine formed was isolated by ion- 30 300 exchange chromatography on Amberlite CG-50 (200 to 400 mesh). 3 Drug Administration. MFMH was delivered continuously from an Alzet 20 200 ?- osmotic minipump (Alza, Palo Alto, Calif.) implanted s.c. in the upper part of the groin facing the tumor leg in rats or in the middle of the back z â€¢* IO 100 in mice (daily doses are described in the table legends; pumping rate Ã• l was 1 Ail/hr). Control animals were implanted similarly with an empty ZC minipump. DFMO was administered p.o. by offering a 2% aqueous solution of the compound in tap water as the sole drinking fluid. Daily IO 15 2O 25 30 drug intake was estimated by measuring the total fluid intake per group DAYS AFTER TUMOR IMPLANTATION of animals. Chart 1. Growth of HTC hepatoma induced i.m. in rats. Changes in tumor ODC and HOC activities and in tumor putrescine and histamine levels with time. Buffalo Materials. Cimetidine (Tagamet) was purchased from Smith Kline & rats were given i.m. injections, in the leg, of 2 x 106 HTC cells. The evolution of French, Puteaux, France. Dexchlorpheniramine malÃ©ate(Polaramine) tumor size was followed with time. Animals were killed at different times, and their was purchased from Laboratoire CÃ©trane,Levallois, France. Compound tumors were removed, homogenized, and analyzed for biochemical parameters. 48/80, ornithine, histidine, and histamine were obtained from Sigma Samples collected up to Day 5 following inoculation are largely contaminated with Chemical Co., St. Louis, Mo. L-[2,5-3H]Histidine (40 to 50 Ci/mmol), S- muscular tissue; thereafter, the tumor can be clearly dissected from the muscle. At adenosyl-L-[mefhy/-3H]methionine (80 Ci/mmol), and DL-[1-'4C]ornithine the end of the experimental period, the tumors weighed 13 Â±2 g. Points, means of 3 animals except for tumor growth studies in which 6 animals were followed; (59 mCi/mmol) were supplied by The Radiochemical Centre, Amersham, oars, S.E.M.

640 CANCER RESEARCH VOL. 44

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Biosynthesis of Histamine, Polyamines, and Cancer maximum at Day 9, and decreased slowly towards a low level

thereafter (Chart 2). No ODC activity was seen in the normal IOO lung, but when mÃ©tastases developed at Day 20 the enzyme activity was markedly increased. The induction of HOC activity 50 in the LL carcinoma paralleled closely that of ODC. The HOC activity in the primary tumor was maximal at Day 8 following inoculation, and the activity in lung was increased at Day 20 when mÃ©tastases were present (Chart 2). Putrescine content " 800 increased rapidly in the tumor associated with the induction of ODC, to reach a maximal level at Day 9 following inoculation. E 400 Spermidine and spermine levels also increased in the tumor, but e with a further delay (Chart 3). The putrescine concentration of O the lungs increased when mÃ©tastaseswere detected. The sper- 1200 midine levels also tended to increase with time, and the spermine â„¢ 800 content of the lungs did not change significantly. The tumor histamine content tended to decrease with tumor growth, and o | 4OO there was no increase in histamine in the lung corresponding to the appearance of mÃ©tastases(Chart 3). O 30

O 5 IO I5 2O DAYS AFTER INOCULATION

GÃŒ Chart 3. LL carcinoma in mice. Changes in polyamine and histamine levels in i the primary tumor and in the lung. Same experiments as in Chart 2. Polyamines U and histamine were measured on acid supematants from tissue homogenates. For further details, see legend to Chart 2.

o Table 1 Effect of cimetidine on the growth of LL carcinoma implanted i.m. in mice 60 section of primary tumor of of the lung Treatment*None (sqcm)4.11 metastases/lung11.4 (9)0.1 Â±0.21" Â±1.6 86 Â±0.008 Cimetidine, -100 mg/ 40 50.023.46 Â±0.1 .30.026.6 Â±1 0.151Â±0.005p kg/daynestCross

O i Effect of Antagonists of Histamine Receptors on the Devel opment of Animal Tumors

75 Cimetidine, a specific antagonist of Hz histamine receptors, o> has been shown to reduce mÃ©tastases in mice bearing LL SO carcinoma (13, 23). We have confirmed the antitumoral effect of cimetidine on LL carcinomas in mice and extend the observations to include HTC tumors in rats. When given at 0.5 g/liter in u drinking water to mice developing LL carcinoma (mean daily drug o I intake, ~100 mg/kg), cimetidine significantly reduced by 17% the growth of the primary tumor, by 42% the number of mÃ©tas 5 IO 15 2O tases, and by 20% the lung mass at Day 20 following inoculation DAYS AFTER TUMOR IMPLANTATION of the tumor in the leg (Table 1). Cimetidine given at 0.5 g/liter Chart 2. Growth of LL carcinoma in mice. Changes in ODC and HOC activities in drinking water (intake, ~50 mg/kg/day) inhibited by 31% the with time in the tumors and in the lungs. Tumors were induced by i.m. injection of 0.5 x 106 3LL cells. The tumor size was measured at various intervals. Animals development of HTC hepatoma in rats [0.02 < p < 0.05 (Chart were sacrificed, and their primary tumor (â€¢)andlungs (O) were removed, homog 4)]- enized, and analyzed biochemically. Points, means of 5 determinations; oars, S.E.M. The reading at Day 0 represents muscle alone; as in Chart 1, samples of tumors Dexchlorpheniramine, a specific antagonist of H! receptors, collected up to Day 6 following inoculation are contaminated with muscle. did not have antitumoral effects on the HTC hepatoma when

FEBRUARY1984 641

administered at 0.025 g/liter (mean intake, 2.5 mg/kg/day). More Antitumoral Effects of MFMH over, when dexchlorpheniramine was combined in therapy with cimetidine (0.5 g/liter in water), it completely abolished the anti- Effects of MFMH and DFMO on the Development of LL tumoral effect of cimetidine (Chart 4). Carcinoma in Mice. When given s.c. by minipump at 100 mg/ kg/day, starting on Day 1 following inoculation with LL carci noma, MFMH inhibited tumor growth by 20% and the number of Effect of Histamine Depletion in the Tumor by Local Injection lung mÃ©tastases by 68% (Table 2). It blocked HOC activity by of Compound 48/80 85% and markedly reduced the histamine content of the tumor The histamine releaser, compound 48/80, was injected locally without significantly affecting the ODC activity and the concen in the tumor-bearing muscle (0.1 mg daily per rat bearing HTC tration of putrescine. DFMO given at 2% in drinking water (mean hepatoma). After 4 local injections of Compound 48/80, the daily intake, approximately 3.4 g/kg from Day 2 of implantation) histamine content of the tumor was reduced from 6.8 Â±0.5 fig/ diminished both the primary tumor size and the number of lung g to 0.5 Â±0.3 ng/g (n = 7). Local treatment with Compound 48/ mÃ©tastases, inhibited ODC, and reduced the tumor putrescine 80 caused only a slight inhibition of tumor growth 21 days after concentration (see also Ref. 1). The activity of HOC and the inoculation (Chart 4). When combined with cimetidine, however, concentration of histamine were slightly although not significantly the antitumoral effect achieved was greater than that seen with increased. When the 2 compounds were combined, all 4 bio cimetidine alone (Chart 4). chemical parameters were markedly reduced, and tumor growth was further decreased to 29% of control for the primary tumor and to 12% of control for the number of mÃ©tastases(Table 2). Effect of MFMH on the Development of EMT6 Sarcoma in Mice. When given s.c. by minipump at 180 mg/kg/day from Day 4 following inoculation of EMT6 cells, MFMH inhibited tumor growth by 40% at Day 17 and reduced the tumor HOC activity z and histamine levels to 13 and 15% of the control values, o respectively, without significantly affecting ODC, SAMDC, or the concentration of putrescine (Table 3). DFMO given at 2% in 2 - drinking water (mean daily intake, approximately 4 g/kg) inhibited

O tumor growth by 47%, reduced the ODC activity and putrescine ce levels by approximately 80%, and increased by 4- to 5-fold the SAMDC activity. HOC was inhibited to a small extent, and there tr o was a 3.5-fold increase in the tumor histamine concentration. S D When both drugs were combined, tumor growth was further reduced to 32% of control (Table 3). However, ODC activity and putrescine levels were decreased less after the combined treat ment than after DFMO alone; similarly, HOC activity and hista DAYS AFTER INOCULATION mine concentrations were also reduced less after combined Chart 4. Effect of histamine receptor antagonists and of Compound 48/80 on treatment than after treatment with MFMH alone. SAMDC activ the growth of HTC hepatoma Â¡nrats. Tumors were inoculated as described in "Materials and Methods." Drug treatment was started 1 day after implantation. A: ity was increased to the same extent after DFMO alone or after â€¢,control tumors; A, cimetidine (C, CIM, CIMET) given at 0.5 g/liter (average daily the combined treatment of DFMO plus MFMH. intake, 50 mg/kg) in drinking water alone; O, cimetidine combined with dexchlor Effect of MFMH and DFMO on the Development of HTC pheniramine (POLA) at 0.025 g/liter in drinking water (intake, 2.5 mg/kg/day); *, dexchlorpheniramine at 0.025 g/liter alone. 8: â€¢control tumors; O, Compound 48/ Hepatoma in Rats. MFMH, given s.c. by minipump at 60 mg/ 80 injected in the tumor-bearing muscle alone; A, Compound 48/80 combined in kg/day starting on Day 1 following inoculation of HTC cells, was therapy with cimetidine given in water. Compound 48/80 was injected locally in the at least as effective as DFMO given at 2% in drinking water from tumor-bearing muscle once daily on 4 consecutive days every week at a dose of 0.1 mg/rat. Biochemical data were measured on samples taken 1 day after the last Day 4 (mean daily intake, approximately 2.5 g/kg) in inhibiting injection. Points, means of 5 rats; oars, S.E.M. the growth of the tumor (Table 4; Chart 5). Combined adminis-

TaWe2 Effect of MFMH and DFMO on LL carcinoma in C57BL mice MFMH was infused s.c. at 100 mg/kg/day from 24-hr after tumor inoculation via an Alzet minipump. DFMO was given as a 2% solution in drinking water from Day 2 following inoculation. Mice were sacrificed on Day 20. Results are presented as percentage of the values obtained from 0.9% NaCI solution-treated animals which were: tumor cross section, 3.5 sq cm; metastatic nodules/lung, 19; ODC, 8.2 nmol/g/hr; putrescine, 20 nmol/g; HOC, 100,000 cpm/g/hr; histamine, 7 nmol/g. Statistical evaluations were by f test. valueTreatment0.9% % of control

size100 mÃ©tastases100 Â±3" NaCI solution Â±12 Â±12 Â±12 Â±29 Â±20 80 Â±4" 32 Â± 36 15 Â± 6" 11 Â±3*116Â±21 MFMH 138 Â±15 92 Â± 6 40Â±2t> 19Â± T6 ^Â±12* 33+8* DFMO 135 Â±30 29 Â±5"Lung 12 Â± 56ODC10054 Â±12"Putrescine10026 Â± 6*HOC1007Â± 4"Histamine10023 Â± 6* MFMD + DFMOTumor a Mean Â±S.E.M.of 5 animals. bp < 0.01 compared to equivalent 0.9% NaCIsolution-treated value.

642 CANCER RESEARCH VOL. 44

Tables Effect of MFMHand DFMOon EMT6sarcoma in BALB/c mice MFMH was infused s.c. at 180 mg/kg/day via an Alzet minipump starting 4 days after s.c. inoculation of BALB/c mice with EMT6 sarcoma cells. DFMO was given as a 2% solution in drinking water from Day 4 following inoculation. Mice were sacrificed at Day 17. Results are presented as percentage of the values obtained from 0.9% NaCI solution-treated animals which were: tumor weight, 5.70 g; ODC, 3.5 nmol/g/hr; SAMDC, 317 nmol/g/hr; putrescine, 71 nmol/g; HOC, 98,000 cpm/g/hr; histamine, 1.1 nmol/g. Statistical evaluations by i test. % of control value Treatment0.9% wt100 NaCIsolution Â±10* Â±22 Â± 9 Â±7 Â±12 60 Â± 96 MFMH 91 Â±23 97 Â±10 104 Â±5 13Â± 3" 15Â±136 53 Â± 9* 17Â± 6" 463Â±416 ^Â±3" DFMO 70 Â± 8 350 Â±46 MFMH + DFMOTumor 32 Â±13"ODC10057 Â±21SAMDC100520 Â±46"Putrescine10039 Â±8*HOC10052 Â± 7*Histamine100Â±1835 Â±18" a Mean Â±S.E.M.of 7 animals. 0p < 0.01 compared to equivalent 0.9% NaCIsolution-treated value.

Table 4 Effect of DFMOand MFMH on HTChepatomainduced in rats MFMH was infused s.c. at 60 mg/kg/day via an Alzet minipump starting 1 day after i.m. inoculation of Buffalo rats with HTC cells. DFMO was given as a 2% solution in drinking water from Day 4 following inoculation. Rats were killed, and the tumors removed at Day 16. Results are presented as percentageof the values in untreated animalswhich were: tumor cross section, 3.5 sq cm; ODC, 7.1 nmol/g/hr; putrescine, 107 nmol/g; HOC,46,000 cpm/g/hr; and histamine, 15 nmol/g. Statistical evaluations by t test. % of control value Treatment0.9% Â±9a NaCIsolution Â±12 7 MFMH 53 Â±96 76 Â±20 120 Â±16 29 Â±17" 37 Â±13* 66 Â±46 45 Â± 5Ã– DFMO 22 Â± 3" 110Â±36 90 Â±20 MFMH 4-DFMOTumor100 18Â±3"ODC10031 Â±10"Putrescine100Â±1245 Â± 9"HOC100Â±1814Â± 7"Histamine100Â±33 Â±19" ' Mean Â±S.E.M.of 6 animals. bp < 0.01 compared to equivalent 0.9% NaCIsolution-treated value.

that seen after 60 mg/kg/day (70% inhibition of tumor growth at Day 16). No greater antitumoral effect was obtained by combin ing MFMH at 120 mg/kg/day with DFMO (2% in drinking water from Day 7) (result not illustrated). MFMH was still very effective when given later in therapy (from Day 13) when the i.m. tumor was already well developed (Chart 5) and the antitumoral effect was associated with decreased histamine levels. These results can be compared with those from a similar experiment where DFMO was given in drinking water starting 7 days after inoculation (Chart 5). Adverse Effects of DFMO and MFMH. None of the treatments used had any marked toxic effects on any model in rats or mice. 7 I5 25 However, the tendency in animals treated with DFMO was to DAYS AFTER INOCULATION gain weight less rapidly than controls. As an example, the Chart 5. Effect of MFMH and of DFMO on the growth of HTC cell solid tumors in rats. Buffalo rats were given i.m. injections of HTC cells on Day 0. â€¢,control average weight gain per animal per week and the water intake rats; O, rats receiving 2% DFMO in drinking water from Day 7; A, rats infused with per day for rats with HTC tumors treated with DFMO and MFMH MFMH (60 mg/kg/day) from Day 13 via Alzet minipumps implanted s.c.; A, rats in the doses specified above were 27 g and 21 ml for control infused with MFMH (60 mg/kg/day) from Day 1. Points, means of 5 animals;bars, S.E.M. (except the 3 first control points which are meansof 10 animals). rats, 26 g and 18 ml for rats receiving MFMH, 4 g and 21 ml for rats receiving DFMO, and 13 g and 17 ml for rats receiving the tration of the compounds produced additive antitumoral effects, combined therapy. The only other effect observed was moderate there being only 18% of residual growth at Day 16 after tumor diarrhea in the majority of rats treated with DFMO, which ap inoculation. MFMH selectively decreased the tumor HOC activity peared to be less after the combined treatment with MFMH plus and histamine content (by 71 and 63%, respectively), while DFMO. DFMO selectively decreased the ODC activity and putrescine content (by 55 and 78%, respectively) (Table 4). After combined Effect of MFMH and Cimetidine on the Proliferation of HTC treatment, HOC activity and histamine levels were depleted to Cells Grown in Spinner Culture an extent similar to that after MFMH alone. This was true also for ODC activity but not for the concentration of putrescine which The antitumoral properties of MFMH could be the result of a was increased following combined treatment when compared to direct effect on the tumor or of an indirect effect via the immune treatment with DFMO alone. When MFMH was infused from Day system. To study the possibility of a direct effect of MFMH on 1 at 120 mg/kg/day, it produced a larger antitumoral effect than the proliferation of tumor cells, the drug was added in vitro to

FEBRUARY 1984 643

Table 5 Cimetidine, the H2 histamine receptor antagonist, had clear Effect of MFMHand cimetidine on the proliferation of HTC cells grown in culture antitumoral effects on LL carcinoma and HTC hepatoma (Table Increasing doses of MFMH or 2 rriM cimetidine were added to the culture 1; Chart 4). A direct effect of cimetidine on the tumoral cell in medium 1 hr after seeding. Results of 3 experiments are presented; 2 generation times were taken into account for calculation of doubling time. these 2 animal tumor models is unlikely. Thus, the proliferation TreatmentControl time(hr)23 of 3LL cells in vitro was unaffected by cimetidine at rriM concen Â±2" tration, and these 3LL cells did not bear histamine receptors as Cimetidine(2 mu) 24 Â±2 judged by the flow cytometric technique (23). Similarly, a direct MFMH (0.01 mm) 35 Â±4 effect of cimetidine on the HTC hepatoma can be ruled out since MFMH (0.1 mm) 40 Â±4 Â±5* MFMH (1 mm)Cell-doubling 46 in our hands HTC cells grown in culture were not affected by Mean Â±S.E.M. cimetidine at HIM concentrations and H2 receptors could not be detected using a sensitive adenylate cyclase assay. Thus, the HTC cells grown in culture. MFMH decreased dose dependently antitumoral effect observed with cimetidine in both our tumor the growth rate of HTC cells (Table 5). The doubling time models is likely to be indirect and mediated by the host immune increased 2-fold in the presence of 1 HIM MFMH with no effect system. on cell viability as estimated by the trypan blue exclusion method. Dexchlorpheniramine, the H, histamine receptor antagonist, In contrast, 2 rriM cimetidine in the culture medium did not had no effect per se on HTC hepatoma (Chart 4) or on LL inhibit the proliferation of HTC cells (Table 5). Furthermore, no carcinoma (data not shown). This observation is consistent with histamine H2 receptors could be detected on HTC cells by the recent finding (6) that stimulation of H! receptors and block measurement of the stimulated adenylate cyclase coupled to the ade of H2receptors appear to play a beneficial role in the defense H2 receptor (the basal adenylate cyclase activity was 0.2 pmol of the host against tumors. Furthermore, and, consistent with of cyclic adenosine 3':5'-monophosphate per min per mg of this concept, administration of the H! antagonist in combination protein) and was not stimulated by 10~5 or 10~4 M histamine or with cimetidine appeared to reverse the beneficial effect of the affected by 1(T4 M cimetidine.3 H2 antagonist in HTC hepatoma. The availability of an irreversible inhibitor of ODC (DFMO) DISCUSSION provided the means to show the role of newly synthesized polyamines in the proliferation of tumor cells (21, 24). Similarly, The parallelism observed between the inductions of HOC and the use of an irreversible inhibitor of HOC has provided useful ODC in our experimental tumor models is similar to that de information concerning the role played by nascent histamine in scribed by Watanabe ef al. (35) in mouse skin painted with a the growth of tumors. tumor promoter. A possible association between high histamine- The present work done on 3 different animal tumor models forming capacity and rapid growth has been proposed (7, 15, demonstrates that MFMH, a selective inhibitor of HOC (12), slows 27). Since the marked increase of ODC activity is well known to the development of certain animal tumors. The tumor growth be associated with rapid growth (16,23), it is possible that some retardation achieved with MFMH was in each case associated tumors form putrescine, some tumors form nascent histamine, with inhibition of HOC and a decrease in the concentration of while some tumors form both biogenic amines which could histamine, an observation consistent with the suggestion that subserve similar roles in rapidly growing tissues. The nascent biosynthesis of histamine is required to maintain a maximal rate histamine formed by inducible HOC can be rapidly metabolized, of proliferation. It should also be noted that MFMH inhibited HOC mainly by diamine oxidase which is elevated in rapidly dividing without affecting the 2 other decarboxylases induced in the cells (4, 5). It represents only a small portion of the total tissue tumors, ODC and SAMDC, which confirms that MFMH is a histamine content which can be significantly increased by the selective inhibitor of HOC (12). presence of macrophages at the tumor periphery. All these Several reports have shown that high ODC activity and a high factors could explain why an increase in tumor histamine does rate of polyamine biosynthesis are associated with rapid cell not necessarily parallel the increase in tumor HOC. [Charts 1 and proliferation and that impairment of putrescine biosynthesis by 3; see also Watanabe ef a/. (34, 35)]. the specific irreversible ODC inhibitor, DFMO, inhibits tumor cell The question arises as to the source of the histamine playing proliferation (1, 2, 21, 25). In confirmation of these data, in our 3 a role in tumor growth. Our experiments show that a massive models, the inhibition of tumor growth obtained with DFMO was local depletion of tissue histamine by injection of Compound 48/ clearly associated with inhibition of ODC and a decreased con 80 in the vicinity of the tumors did not cause a marked antitumoral centration of tumor putrescine while HOC activity and histamine effect (Chart 4). This suggests strongly that histamine stored concentrations were not affected. When DFMO and MFMH were within mast cells either in the tumor or in its vicinity plays no combined in therapy, biochemical effects consistent with inhibi functional role in tumor development and raises the possibility tion of both HOC and ODC were observed, although in some that it is nascent histamine formed within the tissue which plays instances (e.g., EMT6 sarcoma) these changes were quantita the critical role rather than the total histamine content. This tively less pronounced than those seen after the individual drugs. notion receives support from the experiments with MFMH. Thus, Nevertheless, the antitumoral effects observed after combined the antitumoral effects of MFMH were observed even after a therapy were always further enhanced, suggesting that a simul short period of treatment (see, e.g., Chart 5) at which time, taneous depletion of histamine and putrescine in the tumor may because of the slow turnover of histamine in mast cells (26, 29), be more beneficial than a more profound depletion of only one it would be unlikely that significant depletion from this site would of these 2 amines. have occurred. The role and mechanism of action of nascent histamine syn thesized by tumor cells are speculative at present. However, 3 P. Mallorga, personal communication. there are 2 possibilities which fit the majority of facts available.

644 CANCER RESEARCH VOL. 44

The first possibility would reflect an action of histamine on the Lebel, B., and Canu, P. Increased tissue histamine in tumor-bearing mice and immune system (32); by activation of H2receptors on suppressor rats. Br. J. Cancer, 43: 684-688,1981. 8. Burton, K. Conditions and mechanism of the diphenylamine reaction for the cells, histamine would depress the host immune system and estimation of deoxyribonucleic acid. Biochem. J., 62: 315-323, 1956. favor the progression of the tumor. Such an immunosuppressant 9. Cox, C., Mukerjee, S., Jackson, M., and Dey, S. Effect of inhibition of histidine decarboxylase on implantation in the mouse. Contraception, 23: 457-460, role for histamine has been implied previously from experiments 1981. showing that the histamine H2 receptor antagonist cimetidine at 10. Danzin, C., Jung, M. J., Grove, J., and Bey, P. Effect of a-DFMO, an enzyme- a dose similar to the one used in our experiments reduced tumor activated irreversible inhibitor of omithine decarboxylase on polyamine levels in rat tissues. Ufe Sci., 24: 519-524,1979. mÃ©tastases and increased lymphocyte proliferation and cell- 11. Douglas, W. W. Histamine and antihistamines: 5-hydroxytryptamine and an mediated toxicity as a result of inhibiting suppressor cell function tagonists. In: L. S. Goodman and A. Oilman (eds.), The Pharmacological Basis (13, 23). These authors found a slightly more pronounced inhi of Therapeutics, pp. 589-629. New York: Mac Millan, 1975. 12. Garbarg, M., Barbin, E., Rodergas, E., and Schwartz, J. C. Inhibition of bition by cimetidine of LL carcinoma metastatic development in histamine synthesis in brain by 4-fluoromethylhistidine, a new irreversible lung than was evident in our study. However, this was observed inhibitors: in vitro and in vivo studies. J. Neurochem., 35:1045-1052,1980. after early amputation of the tumor-bearing foot, whereas the 13. Gifford, R. R., Ferguson, R. M., and Voss, B. V. Cimetidine reduction of tumor formation in mice. Lancet, 7: 638-639,1981. present experiments did not involve any surgery prior to cimeti 14. Graham, P., Harris, J. M., and Salter, M. R. Histamine formation in psoriatic dine treatment. skin. J. Pharm. Pharmacol., 33: 45P, 1981. 15. Grahn, B., and Rosengren, E. Retardation of protein synthesis in rat tumors The second possibility would involve an intracellular effect of on inhibiting histamine formation. Experientia (Basel), 26: 125-126,1970. histamine on unknown targets, perhaps mimicking putrescine or 16. Hershko, A., and Tomkins, G. M. Studies on the degradation of tyrosine spermidine in rapid cellular growth (21). The cytostatic effect aminotransferase in hepatoma cells in culture. J. Biol. Chem., 246: 710-714, 1971. obtained with MFMH on HTC cells grown in culture (Table 5) 17. Ishikawa, E., Toki, A., Moriyama, T., Matsuoka, Y., Aikawa, T., and Suda, M. favors such an intracellular role since these cells have no hista J. Induction of histidine decarboxylase in tumor-bearing rat. Biochemistry, 68: 347-355,1970. mine receptors on their surface and their growth is not affected 18. JÃ¤nne,J., PosÃ², H., and Raina, A. Polyamines in rapid growth and cancer. by the histamine H2 receptor antagonist cimetidine. Furthermore, Biochim. Biophys. Acta, 473: 241-293,1978. the antitumoral effects achieved in vivo with MFMH were much 19. Kahlson, G., and Rosengren, E. New approaches to the physiology of hista mine. Physiol. Rev., 48:155-196, 1968. more pronounced than those observed with cimetidine at a dose 20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein adequate to block the H2 receptors (23). Taken together, these measurement with the Folin phenol reagent. J. Biol. Chem., 793: 265-275, observations suggest that de now-synthesized histamine has 1951. 21. Mamont, P., Duschesne, M. C., Grove, J., and Bey, P. Anti-proliferative an additional role besides stimulation of suppressor cells via H2 properties of oL-a-difluoromethylornithine in cultured cells. A consequence of receptors. These data are thus in agreement with the suggestion irreversible inhibition of omithine decarboxylase. Biochem. Biophys. Res. Com mun., 87: 58-66, 1978. that high rates of intracellular diamine formation (polyamines and 22. Matsuzaki, S., Suzuki, M., Hamana, K., and Itoh, K. Elevated levels of polya histamine) are essential for optimal rates of tumor growth (14). mines and histamine in adenocarcinomas of the thyroid. J. Clin. Endocrinol. MFMH decreased cell proliferation in vitro without cytotoxicity. Metab., 47:1038-1041,1978. 23. Osband, M. E., Shen, Y., Shlesinger, M., Brown, A., Hamilton, D., Cohen, E., Similarly, cytostatic effects were observed with MFMH in vivo Lavin, P., and McCaffrey, R. Successful tumor immunotherapy with cimetidine without detectable toxicity. Inhibitors of HOC could therefore be in mice. Lancet, 7: 636-638,1981. useful adjuvant agents in cancer chemotherapy, a role which has 24. Prakash, N. J., Schechter, P. J., Mamont, P. S., Grove, J., and Koch-Weser, J. Effect of a-difluoromethylomithine, an enzyme activated irreversible inhibitor been proposed for inhibitors of ODC (1, 2, 23, 25). Combined of omithine decarboxylase on L1210 leukemia in mice. Cancer Res., 38:3059- use of ODC and HOC inhibitors might also be useful in this 3062,1978. respect since the antitumor effects were invariably additive in 25. Prakash, N. J., Schechter, P. J., Mamont, P., Grove, J., Koch-Weser, J., and Sjoerdsrna, A. Inhibition of EMT6 tumor growth by interference with polyamine each of the animal models in the present study. biosynthesis, effect of a-difluoromethylomithine, an irreversible inhibitor of omithine decarboxylase. Life Sci., 26: 181-194,1980. ACKNOWLEDGMENTS 26. Reiman, H. Histamine content in human kidneys with hypemephroid carcinoma. Is there a pathophysiological mechanism? Agents Actions, 77, 454-457,1981. We thank Dr. J. Grove for polyamine analysis, Dr. P. Mallorga for the adenylate 27. Rosengren, E., Henningsson, A., Henningson, S., and Persson, L. Polyamine cyclase and H2 receptors binding experiments, and Dr. F. Gerhart for the synthesis metabolism as related to growth and hormones. Med. Biol., 59: 320-326, of MFMH. We thank Drs. J. Fozard and M. Jung for helpful comments during 1981. preparation of the manuscript; Dr. P. Mamont for culture of HTC and EMT6 cells; 28. Russell, D. H. Polyamines in Normal and Neoplastic Growth. New York: Raven M. Nagy, M. Host, I. Fomieles, and C. Grauffel for technical assistance; and C. Press, 1973. Kratz for typing of the manuscript. 29. Scheinman, P., Lebel, B., Lynch, N. R., Salomon, J. C., Paupe, J. R., and Burtin, C. Histamine levels in blood and other tissues of male and female C3H mice. II. Mice carrying a 3-methylcholanterene induced tumor. Agents Actions, REFERENCES 9: 95-96, 1979. 1. Bartholeyns, J. Treatment of metastatic Lewis lung carcinoma with D,L o- 30. Scolnik, A. J., Rubio, M. C., Comolli, R. R., Colombo, L. L., and Caro, R. A. difluoromethylomithine. Eur. J. Cancer. Clin. Oncol., 79: 567-572,1983. Abnormal histamine metabolism in a transplantable syngeneic spontaneous murine mammary adenocarcinoma. Biomedicine (Paris), 35: 84-87, 1981. 2. Bartholeyns, J., and Koch-Weser, J. Effects of a-difluoromethylornithine alone and combined with Adriamycin or vindesine on L1210 leukemia in mice, EMT6 31. Shaft, R. E., and Beaven, M. A. Increased sensitivity of the enzymatic isotopie solid tumors in mice, and solid tumors induced by injection of hepatoma tissue assay of histamine measurement in plasma and serum. Anal. Biochem., 94: culture cells in rats. Cancer Res., 41: 5158-5161,1981. 425-430,1979. 3. Baudry, M., Mattress, M. P., and Schwartz, J. C. The subcellular localisation 32. Shearer, G. M., Melmon, K. L., and Weinstein, Y. Regulation of antibody response by cells expressing histamine receptors. J. Exp. Med., 736: 1302- of histidine decarboxylase in various regions of rat brain. J. Neurochem., 27: 1301-1309,1978. 1307, 1972. 4. Baylin, S. B. Histaminase (diamine oxydase) activity in human tumors; an 33. Taylor, K., and Snyder, S. H. Isotopic microassay of histamine, histidine expression of mature genome. Proc. Nati. Acad. Sei. U. S. A., 74: 883-887, decarboxylase and histamine methyl transferase in brain tissue. J. Neurochem., 79: 1343-1358,1972. 1981. 5. Baylin, S. B., Beaven, M. A., Engelman, K., and Sjoerdsrna. A. Elevated 34. Watanabe, T., Kitamura, Y., Maeyama, K., Go, S., Yamatodani, A., and Wada, H. Absence of increase of histidine decarboxylase activity in mast cell-deficient histaminase activity in medullary carcinoma of the thyroid gland. N. Engl. J. Med., 283. 1239-1244,1970. w/w mouse embryos before parturition. Proc. Nati. Acad. Sei. U. S. A., 78: 4209-4212,1981. 6. Burtin, C., Scheinmann, P., Salomon, J. C., Lespinats, G., and Canu, P. 35. Watanabe, T., Tagushi, Y., Sasaki, K., Tsuyama, K., and Kitamura, Y. Increase Decrease in tumor growth by injection of histamine or serotonin in fibrosar- in histidine decarboxylase activity in mouse skin after application of the tumor coma-bearing mice. Br. J. Cancer, 45: 54-60,1982. promoter tetradecanoylphorbol acetate. Biochem. Biophys. Res. Commun., 7. Burtin, C., Scheinmann, P., Salomon, J. C., Lespinats, G., Frayssinet, C., 700:427-432, 1981.

FEBRUARY 1984 645

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. Involvement of Histamine in Growth of Mouse and Rat Tumors: Antitumoral Properties of Monofluoromethylhistidine, an Enzyme-activated Irreversible Inhibitor of Histidine Decarboxylase

Jacques Bartholeyns and M. Bouclier

Cancer Res 1984;44:639-645.

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