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PERIOD BIOL

The duration of cell proadhesive effects induced by imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate (NAMI-A), a compound endowed with in vivo antimetastatic properties, was tested in vitro on the human epithelial tumor cell line KB. The intensity of proadhesive effects continues to increase up to 48 to 72 h after NAMI-A withdrawal and declines only after 96 h. The proadhesive effect on cells seeded on fibronectin is greater than on plastic, since it already reaches its maximum after 24 h. This effect suggests a role for integrin activation, which is further stressed by the inhibitory activity of the disintegrin molecule echistatin. The intensity and duration of NAMI-A's proadhesive effects are correlated to cell exposure time and to the rapid release of NAMI-A metabolites in the culture medium in the first 5 min after drug withdrawal. These metabolites are probably neutral species with ruthenium-bound bioligands to allow for the rapid exchange between cells and extracellular medium. These data suggest a long-lasting effect of NAMI-A in biological systems, even at very low concentrations, and stress the low and reversible effects on kidney, where it naturally concentrates. These data on proadhesive effects are, further, relevant for in vivo antimetastatic effects, as this adhesion is associated to cell motility and invasion, which in turn are related to tumor malignancy and metastasis.

Imidazolium trans -imidazoledimethylsulphoxidetrachlororuthenate (NAMI-A) was tested in vitro on the pro-adhesive properties, evaluated as resistance to trypsin treatment, which is a bona fide measure of adhesion strength, of KB and HeLa carcinoma cell lines and on human polymorphonuclear neutrophils (HPMN). NAMI-A increased the pro-adhesive activity of KB cells at 0.001 mM concentration, after few minutes incubation and this effect was not influenced by the vehicle used for cell challenge, neither did it depend on NAMI-A concentration or on temperature. The same effect occurred on HeLa cells at 0.01 mM NAMI-A. This effect, detected at concentrations up to 100 times lower than those necessary to block cells at the G 2 -M premitotic phase of cell cycle, or to inhibit matrix metalloproteinase release or cell invasion, was not related to ruthenium uptake by tumour cells. HeLa cells and healthy HPMN, following short exposure to 0.1 mM NAMI-A, assumed a different shape, with the extrusion of filopodia (HeLa) and of large lamellopodia (HPMN), which increased their interactions with the substrate. This effect was attributed to stabilisation, altered turnover and sensitivity to cytochalasin D of actin filaments. Provided that adhesion is associated with cell motility and invasion, these data suggest that NAMI-A may exert antimetastatic properties at concentrations lower than those observed in the lungs at the end of a conventional intraperitoneal treatment in vivo.

Preliminary studies of in vitro cytostatic activity on less polar fraction of the MeOH extract of the plant Euphorbia nicaeensis All., carried out on KB cells, have evinced a relatively low ability of extract to inhibit cell growth. Successive, five glucocerebrosides were isolated from the cerebroside molecular species obtained from this extract using normal and reversed phase column 'flash-chromatography'. The structures of these cerebrosides were determined on the basis of chemical and spectroscopic evidences. Mass spectrometry of dimethyl disulfide derivatives was useful for the determination of the double-bond positions in the long-chain bases.

Purpose. The uptake of NAMI-A (imidazolium trans-imidazoledimethylsulphoxidetetrachlororuthenate) by KB cells in vitro was compared with the effects of this compound on the cell cycle phase distribution of the cells. Methods. NAMI-A uptake was determined by flameless atomic absorption spectroscopy, and the cell cycle phase distribution was determined by flow cytometry. Results. NAMI-A uptake was proportional to its concentration in the incubation medium. The use of a number of incubation conditions showed that NAMI-A uptake from MEM was independent of the presence of serum and dependent on the presence of amino acids in the incubation medium, and that NAMI-A uptake was markedly higher when the cells were incubated in PBS. The uptake increase observed in PBS did not occur when the cells were kept at 0–4°C, suggesting the presence of active transportation of NAMI-A into cells. In addition, the presence of divalent cations such as Ca2+ and Mg2+, appeared to facilitate NAMI-A uptake. The anionic substance transport inhibitor probenecid significantly reduced the active transportation of NAMI-A into cells. The effects of NAMI-A on cell cycle distribution were strictly dependent on its uptake by tumour cells and not on its extracellular concentration. Conclusions. These findings suggest the interaction of NAMI-A with biological components resulting in possible consequences for the distribution of the compound itself. Furthermore, NAMI-A enters tumour cells both by passive diffusion and by active transportation.

In the context of a research program aimed at elucidating the properties of the 5 H -imidazo[2,1- c ][1,4]benzodiazepine system, a series of 11-aryl-5 H -imidazo[2,1- c ][1,4]benzodiazepines ( 3a – i ) and their 10,11-dihydro-derivatives ( 4a – i ) has been synthesized. The synthetic strategy includes the preparation of the aryl-[1-(2-nitrobenzyl)-1 H -imidazol-2-yl]methanones ( 5a – i ) followed by their reduction and subsequent cyclization. Affinities of compounds 3a – i and 4a – i for central benzodiazepine as well as for adenosine A 1 -receptors were determined by radioligand binding assays. Among the unsaturated analogues, the highest activity at both receptors is displayed by 11-(2-thienyl) derivative 3e . The hydrogenated analogues 4a – i do not exhibit considerable binding affinity either for central benzodiazepine or for adenosine A 1 -receptors.

In PC12 cells, forskolin as well as the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased intracellular adenosine-3',5'-cyclic monophosphate (cyclic AMP) levels, which peaked at 45-60 minutes and declined thereafter. Maximum levels were 3000 and 1700 pmol/10(6) cells during treatment with 10 microM forskolin or 0.1 microM NECA, respectively. Extracellular cyclic AMP rose with time, at mean rates of 24.7 (forskolin) and 11.3 (NECA) pmol/min/10(6) cells. With either drug, a linear correlation was obtained between the calculated time integral of intracellular cyclic AMP and the measured extracellular cyclic AMP levels, indicating that the outflow of cyclic AMP was sustained by a nonsaturated transport system. The ability of forskolin to increase intracellular and extracellular cyclic AMP levels was hindered in a concentration-dependent manner by 8-(p-sulfophenyl)theophylline (8-SPT). A similar inhibition was exerted by other two adenosine receptor antagonists, 8-cyclopentyl-1,3-dipropylxanthine and 3,7-dimethyl-1-propargylxanthine. The concentration-response curve to adenosine was shifted to the right by 25 microM 8-SPT, whereas that of forskolin was shifted downwards. Adenosine deaminase (ADA, EC 3.5.44, 1 U/mL) reduced the intracellular cyclic AMP response to forskolin by 68%, whereas the adenosine transport inhibitor, dipyridamole (10 microM), significantly increased 1 and 10 microM forskolin-dependent cyclic AMP accumulation. Erythro-9-(2-hydroxy-3-nonyl)adenine (10 microM), an inhibitor of ADA, and alpha,beta-methyleneadenosine 5'-diphosphate (100 microM), an inhibitor of ecto-5'-nucleotidase, did not alter forskolin activity. These results demonstrate that a cyclic AMP extrusion system operates in PC12 cells during adenylyl cyclase stimulation by forskolin and that this stimulation involves a synergistic interaction with endogenous adenosine. However, extruded cyclic AMP does not appear to significantly contribute to the formation of the endogenous adenosine pool.