Involvement of Histamine in Growth of Mouse and Rat Tumors
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[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 (21-23°), 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 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1984 American Association for Cancer Research. J. Bartholeyns and M. Bouclier 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 métastases. 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 ±2 (S.E.) métastases.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 ¿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 37°,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.