Free exchange across cells, and echistatin-sensitive membrane target for the metastasis inhibitor NAMI-A (imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate) on KB tumor cells.

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.

Actin-dependent tumour cell adhesion after short-term exposure to the antimetastasis ruthenium complex NAMI-A.

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 biological assay on cerebroside mixture from Euphorbia nicaeensis All. Isolation and structure determination of five glucocerebrosides.

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.

Influence of chemical stability on the activity of the antimetastasis ruthenium compound NAMI-A.

The influence of chemical stability on the antimetastatic ruthenium(III) compound imidazolium trans-imidazoletetrachlorodimethylsulphoxideruthenium(III) (NAMI-A) in aqueous solution was studied both in vitro and in vivo. The loss of dimethyl-sulphoxide (DMSO) ligand from the compound was tested by using a NAMI-A solution acidified with HCl at pH 3.0 and aged for 0, 4, 8 and 24 h prior to intraperitoneal (i.p.) injection into CBA mice bearing advanced MCa mammary carcinoma. The activity of NAMI-A on lung metastases showed no change even after the loss of DMSO ligand from up to 50% of the molecules. The reduction of NAMI-A did not modify the number of KB cells blocked in the S+G2M phases, independent of whether the reduction occurred outside the cells or after loading the cells with the compound prior to treatment with the reductants (ascorbic acid, glutathione or cysteine). In vivo, the complete reduction of NAMI-A with equivalent amounts of ascorbic acid, glutathione or cysteine prior to administration to mice bearing advanced MCa mammary carcinoma was more active than NAMI-A alone. The data show that NAMI-A, although undergoing a series of chemical modifications, maintains its antimetastatic activity in a broad range of experimental conditions.

In vitro cell cycle arrest, in vivo action on solid metastasizing tumors, and host toxicity of the antimetastatic drug NAMI-A and cisplatin.

The effects of NAMI-A (imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate) are compared with cisplatin on tumor cells cultured in vitro at doses of 1 to 100 microM and on tumor metastases in vivo at maximum tolerated doses. Using mouse tumors that metastasize to the lungs, NAMI-A given i.p. for 6 consecutive days at 35 mg/kg/day, was effective independently of the tumor line being treated and of the stage of metastasis growth. Conversely, cisplatin (2 mg/kg/day for 6 days) was as effective as NAMI-A on MCa mammary carcinoma and TS/A adenocarcinoma and less effective than NAMI-A on Lewis lung carcinoma. Cisplatin reduced body weight gain and spleen weight during treatment and was much more toxic than NAMI-A on liver sinusoids, kidney tubules, and lung epithelium. In vitro NAMI-A caused a transient cell cycle arrest of tumor cells in the premitotic G2/M phase, whereas cisplatin caused a progressive dose-dependent disruption of cell cycle phases. Correspondingly, NAMI-A did not modify cell growth, whereas cisplatin caused a dose-dependent reduction of cell proliferation, as determined by sulforhodamine B test. Thus, NAMI-A, unlike cisplatin, is a potent agent for the treatment of solid tumor metastases as well as when these tumor lesions are in an advanced stage of growth. NAMI-A is endowed with a mechanism of action unrelated to direct tumor cell cytotoxicity, and such mechanism of action is responsible for a reduced host toxicity.

Spectroscopic and biological properties of palladium(II) complexes of ethyl 2-quinolylmethylphosphonate.

Spectroscopic (1H NMR, UV-visible) and biological (cytostatic, antiviral activity) studies of palladium(II) complexes of monoethyl 2-quinolymethylphosphonate (2-Hmqmp): dihalide adducts trans-Pd(2-Hmqmp)2X2, chelate Pd(2-mqmp)2.2H2O and ion-pair salt complexes [2-H2mqmp]+[Pd(2-Hmqmp)X3]- (X = Cl, Br), have been carried out in order to determine structural and biological properties of these biologically interesting complex compounds. The complexes were evaluated in vitro for their cytostatic activity against murine L1210 and human KB and T-lymphoblast Molt4/C8 and CEM/0 cell lines, and the results obtained were compared with those obtained for the complexes of diethyl 2-quinolylmethylphosphonate (2-dqmp). The L1210 cell was the most responsive line and complexes of diester 2-dqmp were more active than complexes of monoester 2-Hmqmp. A good relationship was observed between the cytostatic activity of the complexes and their lypophilicity or solubility. Some complexes exhibited significant cell growth inhibitory effects, but none of the them was more cytostatic than cisplatin. Both 2-dqmp and 2-Hmqmp complexes were also evaluated in vitro for their antiviral activity in different assay systems, comprising a broad spectrum of DNA and RNA viruses, but no specific antiviral effects were noted. In addition, the complexes did not show any specific anti-HIV activity.

Palladium(II) complexes of dialkyl alpha-Anilinobenzylphosphonates. Synthesis, characterization, and cytostatic activity.

The new palladium (II) halide complexes with diethyl and dibutyl esters of (alpha-anilino-N-benzyl) phosphonic acid and diethyl and dibutyl esters of [alpha-(4-benzeneazoanilino)-N-benzyl] phosphonic acid have been prepared and studied. All organophosphorus ligands form dihalide complexes, trans-Pd(L)2X2(X = Cl or Br), with monodentate N-bonded ligand through the anilinobenzyl nitrogen in (alpha-anilino-N-benzyl) phosphonate complexes and through the azo nitrogen in [alpha-(4-benzeneazoanilino)-N-benzyl] phosphonate complexes, respectively, without participation of the phosphoryl group. Azobenzene containing ligands by ortho-metallation also form binuclear organo-palladium complexes, [Pd(L-H)Cl]2, with the metal-metal chloro bridge. The complexes have been identified and characterized by elemental analysis, infrared and 1H NMR, as well as by magnetic and conductometric measurements. All were tested in vitro for their cytostatic activity against KB and L1210 tumor cell lines. The results show that these complexes inhibit the multiplication of these tumor cells, but only the dichloro adduct of diethyl [alpha-(4-benzeneazoanilino)-N-benzyl] phosphonate was found to have activity comparable to that of the antitumor drug cisplatin.

Pharmacological and toxicological studies on new Rh(I) organometallic complexes.

New rhodium(I) complexes, belonging to the general structure [Rh(CO)2 (L)], where dithiocarbamate and xanthate derivatives, were synthesized and assayed as cytostatic and antitumour agents in vitro against KB cells and in vivo against P388 leukaemia, Ehrlich ascites carcinoma, Sarcoma 180 ascites and ADJ/PC6A solid tumour. Assays against five trypanosoma strains were also performed. Among the new compounds the [Rh(CO)2 (DPA-dtc)] appeared to be active in all biological systems without showing evident nephrotoxicity.

Synthesis, chemical characterization and biological evaluation of new platinum (II)-sulfonamide complexes.

Six new platinum (II)-sulfonamide complexes were examined for their in vitro cytostatic properties as well as in vivo antitumour effect against three experimental murine tumours. The possible antitrypanosomic in vivo activity against T. brucei, T. congolense and T. cruzi infections was also evaluated. The synthesis and chemical characterization of new complexes is reported. Only two sulfadiazine derivatives appeared to be effective mainly against Ehrlich ascites but in a smaller extent than cisplatin. A satisfactory correlation between antitumour and antitrypanosomic activity was found.

Synthesis and biological evaluation of new Rh (I) complexes with sulfonamide derivatives.

New rhodium(I) complexes, belonging to the general structure [Rh(CO)2(L)], where L were sulfonamide derivatives, were synthesized and characterized by chemical analysis and IR determinations. These complexes were assayed as cytostatic and antitumour agents in vitro against KB cells and in vivo against P388, Ehrich ascites and advanced B16 melanoma. Assays against three Trypanosoma strains were also performed. Among the new compounds, the [Rh(CO)2-(sulfamethoxydiazine)] appeared to be active in all biological systems without showing evident nephrotoxicity. Relationships between biological activity and pi electronic charge localization on N atom of the ligand amidic group were also discussed.

Uptake of adriamycin by rat and mouse mast cells and correlation with histamine release.

It has been shown recently that anthracyclines induce an important, noncytotoxic histamine release from rat and mouse peritoneal mast cells. In the present study we evaluate if histamine release is related to Adriamycin uptake; in addition the uptake of this antineoplastic drug is studied in other normal or tumor cells. In rat and mouse peritoneal mast cells, Adriamycin-induced histamine release is quantitatively related to its intracellular concentrations. Significant differences in Adriamycin uptake are observed among mast cells and other cells. In mast cells, both the uptake of the antineoplastic drug and histamine release are greatly limited by the antiallergic drug sodium cromoglycate; on the contrary, in other normal or neoplastic cells, sodium cromoglycate only slightly reduces Adriamycin intracellular concentrations. When tested on KB cells in culture, sodium cromoglycate does not interfere with Adriamycin cytotoxicity. In mast cells, Adriamycin uptake is temperature dependent, with an optimum at 37 degrees C, and slower than histamine release; histamine release was in fact completed in 30 s, whereas the uptake reached its maximum in 30 min. The influx is pH dependent, with a maximum at pH 6.8, and is blocked by the metabolic inhibitor antimycin A and omission of glucose. It is also possible to limit Adriamycin uptake and histamine release with the calmodulin inhibitors chlorpromazine and trifluoperazine and with the calcium antagonist nicardipine. Adriamycin efflux from mast cells is extremely rapid, temperature dependent (very slow at 0 degrees C), but pH independent, and not modified by metabolic inhibitors. These findings suggest that Adriamycin accumulation in mast cells involves an active transport system which can be inhibited by several agents, among which are sodium cromoglycate, and calmodulin and calcium antagonists.

Algae as possible sources of antitumoural agents. Preliminary evaluation of the "in vitro" cytostatic activity of crude extracts.

Aqueous, ethanolic and chloroformic extracts of 13 algae from the Gulf of Trieste (North Adriatic Sea) were assayed in vitro against human tumour established cell line (KB). Some of them exhibit a noteworthy cytostatic activity, in particular Dictyopteris membranacea (Stackh.) Batt., which significantly inhibits the growth of the cell cultures in all the three different crude extracts, was quantitatively screened in different periods of the year for the presence of cytostatic compound(s).

Cytostatic and antitumour properties of a new series of Pt (II) complexes with cyclopentylamine.

Ten new Pt (II) complexes were synthesized and tested as potential antitumor drugs in vitro on KB human tumour cell line, and in vivo against four experimental tumour systems (P388, L1210, ADJ/PC6A and Yoshida sarcoma). The complexes contained two primary amine ligands (cyclopentylamine) with bidentate leaving ligands consisting of nitro-, dinitro- and sulfo-derivatives of phthalic and isophthalic anions. Various complexes showed a good cytostatic effect in vitro with ID50 from 0.29 and 0.99 mcg/ml. The Pt(cpa)2 (5-sulfo-IPA) appeared to be the most effective compound against P388 and L1210 (T/C% 310 and 250 respectively after three 50 mg/Kg i.p. injections) as well as against ADJ/PC6A (ID90 2.8 mg/kg after a single i.p. injection) and Yoshida sarcoma (T/C% 17.5 after a single 50 mg/kg i.p. injection), but the phthalic acid nitro-derivatives were also quite effective. As far as antileukemic effect was concerned, there was a fairly good correlation between the results in vitro and in vivo. Relationships between antitumour activity and pi electronic charge localization on O- atoms of leaving ligands (M.O. Huckel's Calculations) are also discussed.

Synthesis and in vitro cytotoxic activity of semisynthetic derivatives in the santonin series.

The synthesis of two new santonin derivatives namely 3-oxo-6 beta-H-11 beta-phenylselenoeudesm-1,4-dien-6,13-olide and 3-oxo-6 beta H-eudesm-1,4,11-trien-6,13-olide is reported along with the results of a series of santonins tested for activity against the growth of KB cells in vitro, a human epidermoid nasopharynx carcinoma. Select compounds were found to be active at concentrations lower than 5 X 10(-5) M. In particular, the compound 2 alpha-bromo-3 beta-hydroxy-6 beta H-eudesm-11-en-6,13-olide exhibits an extremely low ID50 value at 0.33 X 10(-6) M. Some relationships between chemical structure and cytotoxic activity are suggested, i.e. the alpha-methylene-gamma-lactone moiety appears to be necessary for cytotoxic activity toward KB cells growth in vitro by santonin derivatives.

In vitro cytostatic activity of chromatographic fractions of extracts from some species of compositae.

Three species of the family of Compositae were extracted, partitioned and fractioned on silica gel column. The chromatographic fractions were investigated for cytostatic activity in vitro against human KB (human carcinoma of nasopharynx) cell line. The most interesting results were obtained with the first eight fractions of Senecio inaequidens DC, chloroformic extract, which showed marked cytostatic activity and appeared worthy of further investigations.

New cytotoxic guaianolides and derivatives from Grosheimia macrocephala.

Grosheimia macrocephala (Compositae) proved to be a very interesting natural source, not only of grosheimin (I), but also of cynaropicrin (III). Some derivatives from these two natural sesquiterpene gamma-lactones were prepared (Schemes 2 and 3) and two new natural guaianolides: 4'-deoxy-cynaropicrin (V) and 4'-nor-2'-metoxy-cynaropicrin (VI) were isolated and structurally identified. A series of these compounds were submitted to in vitro preliminary testing of cytostatic activity against KB cell lines and relationships between bioactivity and chemical features were investigated.

On the synthesis, cytostatic and antitumor properties of new Pt(II) and Pt(IV) complexes with chloroanilines.

The paper reports the synthesis, the chemical characterization and the IR data of new Pt(II) and Pt(IV) complexes, as well as their cytostatic activities on KB cells and antitumour properties against three tumour systems (P388 and L1210 leukemias and advanced B16 melanoma). The following ligands were used: 2,5-dichloroaniline, 3,4-dichloroaniline, 2,4,6-trichloroaniline, 3,4,5-trichloroaniline, 2,3,4,5-tetrachloroaniline and 2,3,5,6-tetrachloroaniline. The tri- and tetrachloroaniline-Pt(II) complexes displayed a fairly good antileukemic activity but lower than cisplatin. The effect of these compounds against advanced B16 melanoma appears more interesting. They show an activity comparable or in some cases higher than cisplatin and other 1,2-diaminocyclohexane-Pt(II) complexes. The M.O. Huckel's calculations were performed on the ligand molecules in order to help us to draw a structure-activity relationship for new compounds.