Comparison of antiproliferative effects of 1-histamine-2 receptor antagonists,cimetidine, ranitidine,and famotidine,in gastric cancer cells

In the immune system, histamine is known to suppress cytotoxic T-lymphocytes and mitogen induced lymphocyte thymidine uptake, down-regulate some cytokines, and activate suppressor T-lymphocytes, and in the gastrointestinal system, histamine was reported to have trophic effects on gastrointestinal epithelial cells. Enhanced rates of cell proliferation by histamine are implicated in the pathogenesis of carcinogenesis. This study was designed since there is a lack of comparative data about the cell proliferations of histamine-2 receptor antagonist (H2-RA), cimetidine, ranitidine, and famotidine, in gastric cancer. KATO-III and AGS cell lines were used in this experiment. The concentrations of the histamine and cimetidine were 10⁻⁵, 10⁻⁸M, respectively and those of ranitidine and famotidine were 10⁻⁶-10⁻⁹M, respectively. Cell proliferation after drug treatment was evaluated by direct cell counting, [³H]thymidine incorporation, and MTT assay. Activities of ornithine decarboxylase (ODC), a rate limiting enzyme in polyamine synthesis, were measured after each drug treatment. Protein kinase A, a cAMP-dependent protein kinase system, was assayed using [α-³²p]ATP. Histamine showed statistically significant cell proliferating effects in a dose-dependent manner (P < 0.01), the maximal effect in 10⁻⁵M concentration. ODC activities were increased in accordance with the increment of cell numbers after histamine treatment. Cimetidine reversed the histamine-stimulated cell proliferation significantly, the maximal effect in 10⁻⁵M concentration (P < 0.01). Although ranitidine showed the tendency to attenuate the cell proliferation dose-dependently, but without statistical significance, famotidine did not show such an effect at all. cAMP-dependent protein kinase activities were significantly increased following 10⁻⁵M histamine treatment, also reversed significantly by cimetidine co-administration (P < 0.01). Beneficial clinical outcomes could be anticipated from cimetidine treatment in patients with gastric cancer by anti-proliferating effects against gastric cancer cells. These effects of H2-RA are likely to be mediated by specific interactions at the H2-receptor.     

Responses to histamine and selective H2-receptor agonists in lung parenchymal strips from normal and sensitized guinea-pigs

Histamine produces concentration-dependent contractions of lung parenchyma strips obtained from normal and sensitized guinea-pigs. The responsiveness of the sensitized lung strips to histamine was significantly increased compared to normal tissues. Clemizole (0.1 M) was equally effective as an H₁-antagonist in normal (dose-ratio 9.12) and sensitized (dose-ratio 9.77) tissues.The concentration-response curves to histamine were displaced to the left by cimetidine (0.1 M to 0.1 mM) with similar dose-ratios in normal and sensitized tissues. Cimetidine enhanced maximal responses to histamine only in normal lung strips. The effects of submaximal equieffective concentrations of histamine were augmented to the same extent by cimetidine (0.1 mM) in normal and sensitized tissues. The responses to histamine were not modified by indomethacin (5 M).The responsiveness and sensitivity of sensitized lung strips to isoprenaline, impromidine, 4-methylhistamine and dimaprit were not different from those of normal tissues. Cimetidine yielded, as antagonist of dimaprit, similar pA₂ values in normal and sensitized tissues.In conclusion, there is no experimental evidence in favour of the existence of an impairment of H₂-receptor activity in sensitized airways. Hyperreactivity to histamine is probably due to differences between normal and sensitized tissues with respect to Ca²⁺ entry and/or intracellular Ca²⁺ release in response to H₁-receptor activation.     

The influence of Cimetidine and Ranitidine on basophil histamine release and bronchial reactivity in asthmatic patients

Anti-IgE induced histamine release from isolated basophils after Cimetidine and Ranitidine administration was evaluated in 22 patients with atopic bronchial asthma.The histamine provocation test after Ranitidine treatment in 10 patients with atopic bronchial asthma and 10 patients with peptic ulcer was also performed.Investigationsin vitro revealed that Cimetidine and Ranitidine in low concentrations had an inhibitory effect whereas in concentrations of over 10^–6 M and 10^–4 M, respectively, they enhanced histamine release.Investigationsin vivo after administration of Ranitidine showed that it does not cause marked changes in the bronchial reactivity in patients with bronchial asthma and any change in patients with peptic ulcer.These preliminary studies seem to suggest that in patients with atopic bronchial asthma and concomitant peptic ulcer Ranitidine is preferable to Cimetidine in the treatment of digestive tract disorders.This work was supported by the Polish Academy of Science (grant Nr 06.03).     

Enhancement of neutrophil infiltration in histidine decarboxylase-deficient mice

The roles of histamine in the anaphylactic increase in vascular permeability and leucocyte infiltration were analysed in an air pouch-type allergic inflammation model in histidine decarboxylase-deficient (HDC^−/−) mice and wild-type mice. In the immunized wild-type mice, histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase were increased by injection of the antigen solution into the air pouch. However, in the immunized HDC^−/− mice, the antigen challenge did not increase histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase. Number of leucocytes (more than 83% are neutrophils) in the pouch fluid 4–24 hr after the antigen challenge in the HDC^−/− mice was significantly higher than that in the wild-type mice. Simultaneous injection of histamine with the antigen solution into the air pouch of the immunized HDC^−/− mice reduced the antigen-induced leucocyte infiltration at 4 hr. Simultaneous injection of the H2 antagonist cimetidine but not the H1 antagonist pyrilamine with the antigen solution into the air pouch of the immunized wild-type mice further increased leucocyte infiltration at 4 hr. The levels of macrophage inflammatory protein-2 at 2 hr and of tumour necrosis factor-α at 4 hr in the pouch fluid of the HDC^−/− mice were significantly higher than those of the wild-type mice. These findings indicate that histamine plays significant roles not only in the anaphylactic increase in vascular permeability via H1 receptors but also in the negative regulation of neutrophil infiltration via H2 receptors in allergic inflammation.     

Effect of histamine on the mitogenic response of human lymphocytes and its modification by cimetidine and levamisole

The phytohaemagglutinin (PHA) mitogenic response of human blood lymphocytes demonstrated a dose-dependent inhibition by histamine (in the range 5×10^–7 M). Using suboptimal mitogenic doses of PHA the suppression was more pronounced. Stimulation using different doses of pokeweed mitogen (PWM) was not altered by histamine. Purified T cells and a lymphocyte population depleted of histamine-receptor-bearing cells were less sensitive to the histamine effect. In the supernatants of histamine-stimulated cultures, suppressor factor activity was found. Cimetidine could reverse the effect of histamine in a dose-dependent manner. Adding levamisole to the PHA cultures also produced some antihistaminic effect.     

Inhibition of suppressor cell function by cimetidine in a murine model

Immunomodulatory effects of cimetidine, an H2 histamine receptor antagonist, have been reported in humans and animals. To define these effects more clearly, the action of cimetidine on suppressor cell function was studied utilizing a murine model of contact hypersensitivity. Intravenous inoculation of BALB/c mice with DNP-coupled syngeneic spleen cells induced the production of DNP-specific suppressor cells which could easily be demonstrated by a reduction in ear swelling after contact sensitization with 1-fluoro-2,4-dinitrobenzene (DNFB) following transfer of spleen and lymph node cells to naive syngeneic recipients. Cimetidine treatment of animals in which suppressor cells were induced resulted in an inability of these mice to transfer cellular suppression as measured by development of a normal immunologic response in the recipient mice. The effect of cimetidine was both dose and time related. While all groups receiving cimetidine showed some loss of suppressor cell function, the maximum effect (up to 100% inhibition) was seen when 50 mg/kg of cimetidine was administered intraperitoneally 2 days before or on the day of suppressor cell induction. Some restoration also occurred when cimetidine was given after the day of induction. It has been shown that suppressor cells possess histamine receptors which may be involved in suppressor cell activation. The results indicate that cimetidine may inhibit the functioning of these receptors.     

Prevention of acid aspiration by H2-receptor antagonists

Summary Cimetidine, a histamine H₂-receptor-antagonist, was administered either as a single 400 mg dose perorally 10–12 h before operation, or in a 400 mg dose perorally 10–12 h before operation plus 200 mg intravenously 1–2 h before operation, in 63 patients awaiting general elective surgery. Distribution of patients showed a significantly greater number of patients with a pH >2.5 in cimetidine groups, as compared to controls only, if the whole time of anaesthesia is taken into consideration. Between the two cimetidine regimens there was no statistically significant difference. These findings suggest that cimetidine is not necessary as a routine pre-operative medication in general elective surgery.     

Bronchial hyper-reactivity in atopic patients during H2-receptor blocker treatment

Bronchial reactivity after Cimetidine treatment was evaluated by the histamine provocation test in 24 patients with atopic bronchial asthma and 10 patients with peptic ulcer. Anti-IgE induced histamine release from isolated basophils was also investigated. After blockade of H₂ receptors, an increase of bronchial reactivity and an exacerbation of clinical symptoms were observed in 4 asthmatic patients [17%]. A moderate increase in bronchial reactivity without exacerbation of asthma symptoms was observed in 16 other patients [66%]. In some of the patients treated with Cimetidine an enhancement of IgE-induced histamine release from isolated basophils was observed.     

A T-dependent factor in mouse serum suppressing lymphocytic anti-tumour cytotoxicity

In a microcytotoxicity assay composed of pooled, urethane-induced lung adenoma cells from BALB/c mice and syngeneic lymphocytes sensitized in vivo to these tumour cells, the addition of serum either from adenoma-bearing or normal BALB/c mice resulted in a depression of the cytotoxic response. This suppressive factor was stable to heating at 56° for 30 min and was unaffected by freezing and thawing. Pre-treatment of tumour targets with serum prior to the addition of lymphocytes was not protective, but pretreatment of sensitized lymphocytes with serum inhibited their cytotoxic ability to the same extent as did treatment of the whole culture with serum. Suppressive activity could be removed from serum by absorption with sensitized, but not with unsensitized lymphocytes. While sera from syngeneic or adenoma-bearing nude mice were free of suppressive activity, the activity appeared in nude animals following thymus implantation.     

Therapeutic effect of cimetidine on acetaminophen-induced hepatotoxicity in rabbits

Acetaminophen is an analgesic and antipyretic drug that may cause hepatic toxicity in humans and experimental animals. Cimetidine is an H₂ blocker used for suppression of gastric acid secretion. One of the side effects of cimetidine is blockade of the cytochrome P-450 enzyme system which results in increased half-life of some drug. In this study, 120 female rabbits, randomized into 12 groups (three control and nine test groups), were used. Acetaminophen, 3.24 g/kg, in suspension form as the LD₅₀, was administered orally to induce liver necrosis. Cimetidine (40 mg/kg) was administered intravenously at 0, 2, and 4 h after administration of acetaminophen. Some treatment groups received cimetidine in two equal divided doses—20 mg/kg cimetidine was administered at 2 and 12 h, 2 and 24 h, 4 and 12 h, and 4 and 24 h after administration of acetaminophen. Blood samples were collected at 0, 12, 24, and 36 h after induction of acetaminophen toxicity. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, bilirubin, and arginase were measured in all groups and were found to be increased in acetaminophen control group and some treatment groups (p < 0.05). Results showed that the best treatment effect of cimetidine could be obtained with whole dose of cimetidine administration and 2 h after acetaminophen intake.     

In vivo effect of cimetidine on canine pulmonary responsiveness to aerosol histamine

A series of experiments were designed to discover whether pulmonary histamine H₂ receptors might be of physiologic importance in vivo in the dog. Dose-response curves were performed to aerosol histamine in 11 dogs both before and 1 hr after H₂ receptor blockade with cimetidine (1 mg/kg as a rapid intravenous infusion). Cimetidine had no significant effect on control values of dynamic compliance or resistance of the lung. In the 11 dogs tested H₂ receptor antagonism significantly potentiated (p < 0.05) the animals' pulmonary responsiveness to aerosol histamine. The potentiation of histamine constrictor effects produced by cimetidine were more marked on those dogs initially least responsive to aerosol histamine (p < 0.01). We have found evidence for the presence of inhibitory H₂ receptors in canine airways and for the distribution of these receptors among dogs, explaining in part the previously described differences among dogs in the pulmonary responsiveness to aerosol histamine.     

Binding of [3H]cimetidine to rat brain tissue

Binding of [³H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22±0.05 M found. This binding is inhibited by a range of imidazole-derived histamine H₂-receptor antagonists, but not by a number of non-imidazole H₂-receptor antagonists. It is concluded that the [³H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H₂-receptor.     

Mechanism of action of H2-antagonists on histamine-or dimaprit-stimulated H2-receptors of spontaneously beating guinea-pig atrium

Cimetidine, ranitidine and famotidine are antagonists of the histamine H₂-receptors on the spontaneously beating right atrium of the guinea pig. When analyzed by the classical Schild method theirpA ₂-values are respectively: 6.3, 6.8 and 7.7 with dimaprit as agonist and 5.8, 6.5 and 7.7 with histamine as agonist. Radiolignad-displacement studies with [³H]-tiotidine as radioligand resulted inpK _d values for cimetidine, ranitidine and famotidine of 6.3; 6.9 and 8.2 respectively.In dimaprit-stimulated atria all antagonists added at concentrations above theirK _d values depressed the maximal increase in frequency. In the presence of histamine this effect was much less pronounced and only visible at concentrations of ranitidine and famotidine around 10 ·K _d.The rightward shift of the curves as well as the decrease inE _max are reversible but the dissociation constants of the antagonists are small (less than 10^–3 s^–1).The spontaneously beating right atrium showed receptor reserve for histamine and virtually no receptor reserve for dimaprit.The results have been interpreted in a model in which H₂-antagonists act mainly by competing with the agonist for the histamine receptor site but have in addition a distinct affinity for a secondary site on the receptor. Occupation of this site by the antagonist prevents building up of the stimulus elicited by the agonist and thus decreases theE _max. In systems with receptor reserve (histamine) the effect of antagonist binding to the secondary binding site is seen only at high concentration of antagonist while in absence of receptor reserve (dimaprit) the depression ofE _max is directly visible.Simulations of the model show that the affinity of this secondary binding site is 50- (famotidine) to 100-(cimetidine and ranitidine) fold lower than for the agonist binding site.     

Histamine H1- and muscarinic receptor antagonist activity of cimetidine and tiotidine in the guinea pig isolated ileum

The histamine H₂-receptor antagonists cimetidine and tiotidine were compared as antagonists for H₁- and muscarinic receptors in the guinea pig isolated ileum. Their interactions with histamine were evaluated on the phasic and tonic components of the histamine response. For carbachol, only the phasic component of the response was monitored.For their interactions with histamine, the type of antagonism, surmountable or nonsurmountable, depended upon the choice of response metameter. Evaluation of the antagonism produced by either tiotidine or cimetidine for histamine concentration-response curves based on the phasic response metameter showed an initial dextral shift with surmountable antagonism. With increasing antagonist concentrations the concentration-response curves were further shifted to the right and the maximum response was depressed. When this antagonism was evaluated for histamine concentration-response curves based on the tonic response metameter, cimetidine produced dextral shifts and nonsurmountable antagonism, and tiotidine exhibited only nonsurmountable antagonist activity. pA₂ values for cimetidine antagonism of the phasic and tonic components of the histamine response were 3.2 and 3.7 respectively, and pD₂ values for the phasic and tonic components were 1.8 and 2.3 respectively. The pA₂ value for tiotidine antagonism of the phasic component was 3.9 and the pD₂ values for the phasic and tonic components were 2.6 and 3.3 respectively.Cimetidine and tiotidine at concentrations which did not depress the phasic component of the histamine response produced surmountable antagonism to the contractile action of carbachol. The pA₂ values for cimetidine and tiotidine antagonism of carbachol were 3.8 and 3.6 respectively.     

Effect ofin vivo antiestrogen pretreatment on rabbit atrial chronotropic response to histamine

The chronotropic response ( rate) to histamine (1.4 to 1.8×10^–6 M) of isolated atria from antiestrogen (tamoxifen)-pretreated immature female rabbit was investigated. Tamoxifen treatment (1.0 and 10.0 mg/kg/day for 14 days) had no significant effect on the rate. The R_max and D_1/2max were not significantly different in the two tamoxifen-treated groups compared to the oil-treated (1.0 ml/kg/day for 14 days) control group. Cimetidine (2.8×10^–7 M) inhibited the rate to histamine in all groups: control, 27%; tamoxifen (1.0 mg/kg), 38%; and tamoxifen (10.0 mg/kg), 28%. Only the low dose of tamoxifen was found to be estrogenic (uterotropic). We conclude that tamoxifen pretreatment, both at estrogen-agonist and estrogen-antagonist doses, is without effect on atrial chronotropic response to histamine.     

Effect of histamine on the hippocampal neurons in guinea-pigs

Histamine produced an excitatory effect on CA3 pyramidal cells in hippocampal slices of guinea-pigs. The amplitude of population spikes and EPSPs was augmented by histamine at concentrations of 10^–7 to 10^–6 M. Cimetidine but not pyrilamine prevented the stimulatory effect. The histamine effect was mimicked by dimaprit but not by 2-pyridylethylamine. It is concluded that histamine has a facilitatory influence on the hippocampal excitatory system via H₂ receptor.     

Increased apoptosis in osteoclasts and decreased RANKL immunoexpression in periodontium of cimetidine‐treated rats

It has been demonstrated that histamine interferes with the recruitment, formation and activity of osteoclasts via H₁‐ and H₂‐receptors. Cimetidine is a H₂‐receptor antagonist used for treatment of gastric ulcers that seems to prevent bone resorption. In this study, a possible cimetidine interference was investigated in the number of alveolar bone osteoclasts. The incidence of osteoclast apoptosis and immunoexpression of RANKL (receptor activator of nuclear factor κB ligand) was also evaluated. Adult male rats were treated with 100 mg kg^?1 of cimetidine for 50 days (CimG); the sham group (SG) received saline. Maxillary fragments containing the first molars and alveolar bone were fixed, decalcified and embedded in paraffin. The sections were stained by H&E or submitted to tartrate‐resistant acid phosphatase (TRAP) method. TUNEL (terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling) method and immunohistochemical reactions for detecting caspase‐3 and RANKL were performed. The number of TRAP‐positive osteoclasts, the frequency of apoptotic osteoclasts and the numerical density of RANKL‐positive cells were obtained. Osteoclast death by apoptosis was confirmed by transmission electron microscopy (TEM). In CimG, TRAP‐positive osteoclasts with TUNEL‐positive nuclei and caspase‐3‐immunolabeled osteoclasts were found. A significant reduction in the number of TRAP‐positive osteoclasts and a high frequency of apoptotic osteoclasts were observed in CimG. Under TEM, detached osteoclasts from the bone surface showed typical features of apoptosis. Moreover, a significant reduction in the numerical density of RANKL‐positive cells was observed in CimG. The significant reduction in the number of osteoclasts may be due to cimetidine‐induced osteoclast apoptosis. However, RANKL immunoexpression reduction also suggests a possible interference of cimetidine treatment in the osteoclastogenesis.     

Immunomodulation of cimetidine in healthy volunteers

Summary The effect of cimetidine, a histamine H2-receptor antagonist, on the immune system in man was investigated in 11 healthy volunteers. Cimetidine was administered orally in daily doses of 800 mg for a period of 7 days. At the end of the administration period the number of peripheral CD8+ (cytotoxic/suppressor) cells had diminished significantly (P<0.05) along with a corresponding increase in the CD4+ (helper/inducer): CD8+ (cytotoxic/suppressor) cell ratio (P<0.01). Compared with pretreatment values, a significant in vitro blastogenic response to mitogen stimulation with concanavalin A (P<0.005), phytohemagglutinin (P<0.01), and pokeweed mitogen (P<0.05) was observed in lymphocytes of volunteers after cimetidine intake. The cell-mediated hypersensitivity as assessed by skin testing of seven recall antigens was also enhanced significantly (P<0.001). Using Spearman's coefficient of correlation to compare mitogen-stimulation tests and skin tests of delayed hypersensitivity to the CD4+:CD8+ ratio, yielded a positive correlation (r=0.89;r=0.85, respectively). These effects were reversible 96 h after the last cimetidine dose. In contrast, leukocytes, total T lymphocytes (CD2+, CD3+), CD4+ (helper/inducer) cells, natural killer cells (Leu7+), immunoglobulins, and total complement, C3, C4 were unaffected by cimetidine administration.Abbreviations Con A concanavalin A - NK natural killer - PHA phytohemagglutinin - PWM pokeweed mitogen This work is dedicated to the memory of Professor Dr. med. E.E. Ohnhaus, a stimulating teacher and a great person, who passed away in 1988     

Glycosylation patterns of the human colon cancer cell line HT-29 detected byHelix pomatia agglutinin and other lectins in culture,in primary tumours and in metastases in SCID mice

Human colonic cancer cells (HT-29, 10 ⁷ cells/dose) were injected subcutaneously between the scapulae of 19 severe combined immunodeficient (SCID) mice. After 19 days, large tumours had developed in 18 out of the 19 animals and the mice were then killed. Metastases were detected in the lungs of 16 animals but not in other organs investigated. Surgical removal of the primary tumour in another group of five animals led to a prolonged survival and further growth of metastases in the lungs. HT-29 injection into the tail vein (n=5)resulted in colonization of the lungs. The tumours that developed in the animals were signet cell carcinomas; these forms are not seen in HT-29 cells in culture. Glycoconjugate expression of the tumours was assessed using several lectins. In many cases the results indicated a stability of lectin-binding patterns from cell culture conditions to implantation into the SCID mice. This was true for the lectin Helix pomatia agglutinin (HPA), the binding of which is associated with a high metastatic potential in some human tumours, including colon cancer. All the primary tumours and metastases were HPA positive. This xenograft tumour model seems to be a clinically relevant system for the study of glycoconjugate expression in human colon cancer cells and their metastases.     

Inhibition of renal alkaline phosphatase by cimetidine

Alkaline phosphatase (ALP) belongs to hydrolase group of enzymes. It is responsible for removing phosphate groups from many types of molecules, including nucleotides and proteins. Cimetidine (trade name Tagamet) is an antagonist of histamine H2-receptor that inhibits the production of gastric acid. Cimetidine is used for the treatment of gastrointestinal diseases. In this study the inhibitory effect of cimetidine on mouse renal ALP activity was investigated. Our results showed that cimetidine can inhibit ALP by uncompetitive inhibition. In the absence of inhibitor the V(max) and K(m) of the enzyme were found to be 13.7 mmol/mg prot.min and 0.25 mM, respectively. Both the Vmax and Km of the enzyme decreased with increasing cimetidine concentrations (0- 1.2 mM). The Ki and IC(50) of cimetidine were determined to be about 0.5 mM and 0.52 mM, respectively.