Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The need for antitumor drugs active on malignant solid tumors by mechanisms unrelated to cytotoxicity, which always encompasses dose- and effectiveness-limiting host toxicity, prompted the development of drugs, such as matrix metalloproteinase inhibitors (Sledge et al., 1995; Yoneda et al., 1997) and antiangiogenic agents (Nagabuchi et al., 1997; Kotoh et al., 1999), active on targets different from the classical DNA and cell division mechanisms. The last decade also showed the development of some classes of ruthenium complexes endowed with interesting chemical properties (Keppler et al., 1987; Clarke 1989). Some of them, such as those characterized by sulfoxide ligands (Mestroni et al., 1989; Sava and Bergamo, 1997), showed a pronounced antitumor activity in experimental models of murine tumors. One of these complexes, namely, imidazolium trans-imidazoledimethylsulfoxide-tetrachloro ruthenate (NAMI-A), evidenced a selective action against lung metastases of solid experimental tumors, irrespective of the lack of a significant reduction of primary tumor growth (Sava et al., 1998, 1999a). NAMI-A, similarly to its predecessor NAMI and unlike cytotoxic anticancer drugs such as Adriamycin and cisplatin, showed no direct cytotoxicity for tumor cells in vitro (Sava et al., 1995; Capozzi et al., 1998; Bergamo et al., 1999). The absence of cell cytotoxicity might explain the apparent lack of host toxicity of this compound. The pharmacological characterization of the favorable ratio between antimetastasis action and host toxicity of NAMI-A has recently prompted the start of a phase I clinical trial as an antimetastasis agent at the Netherlands Cancer Institute of Amsterdam.

The preclinical studies on NAMI-A might then serve to evaluate the nature and weight of the in vitro characteristics that would address new compounds to in vivo studies with high probability to highlight antimetastasis activity even better than that of NAMI-A itself. We therefore thought it worthwhile to compare the effects of four ruthenium complexes in vivo on the solid tumor Lewis lung carcinoma after a careful examination of the in vitro effects that have characterized the pharmacological properties of NAMI-A. The complexes chosen include trans-dichlorotetrakisdimethylsulfoxide ruthenium(II) (trans-Ru) (Sava et al., 1989), originated from the studies on the cis-isomer (Mestroni et al., 1989), which provided a strong evidence of the role of sulfoxide ligands for metastasis reduction; imidazolium trans-imidazoletetrachlororuthenate (ICR) (Keppler et al., 1987), a ruthenium(III) complex that showed activity on a colorectal, chemically induced tumor of the rat (Keppler and Rupp, 1986); and sodium trans-tetramethylensulfoxideisoquinolinetetrachlororuthenate (TEQU) (Sava et al., 1995; Capozzi et al.,1998), a highly liposoluble ruthenium(III), that evidenced a marked tumor cell cytotoxicity, not different from that of cisplatin. The in vitro study was carried out on some common tumor cell lines such as the human-derived MCF-7, LoVo, and KB, and the murine TS/A adenocarcinoma cell lines. The study focuses onto relative antimetastatic potency balanced with the role of in vitro effects and with the ruthenium uptake by tumor cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Compounds and Treatment

NAMI-A (Mestroni et al., 1998), TEQU (Alessio et al., 1993), trans-Ru (Alessio et al., 1988), and ICR (Keppler et al., 1987) were prepared according to already reported procedures. The dose required for in vivo daily administration was dissolved in isotonic nonpyrogenic physiological saline. For in vitro studies NAMI-A, TEQU, trans-Ru, and ICR were dissolved in PBS-Ca²⁺-Mg²⁺ and sterilized by filtration with a 0.2-µm filter. NAMI-A, TEQU, trans-Ru, and ICR were given to mice by i.p. administrations of 35 mg/kg/day (76 µmol/kg/day), 17.5 mg/kg/day (35 µmol/kg/day), 35 mg/kg/day (72 µmol/kg/day), and 70 mg/kg/day (154 µmol/kg/day), respectively, in volumes of 0.1 ml/10 g of body weight. The dose used for each compound may be considered the maximum tolerated and equitoxic dose, corresponding to the LD_0.05-0.1 obtained with the same treatment schedule and route of administration in separate tests with healthy CBA mice. The treatment was performed on days 8 to 13 after tumor implantation.

Tumor Line

Lewis lung carcinoma was originally provided by the National Cancer Institute, Bethesda, MD, and was maintained in C57BL/6 mice (Harlan, S. Pietro al Natisone, Italy) by subcutaneous injection in the axillary region of 10⁶ tumor cells of a single cell suspension, prepared from mincing with scissors the primary tumor masses obtained from donors similarly implanted 2 weeks before (Geran et al., 1972). For experimental purposes, the tumor was propagated in BD2F1 mice (Harlan) by i.m. implantation into the calf of the left hind leg.

Primary Tumor Growth and Lung Metastasis Evaluation

Primary tumor growth was determined by calliper measurements, by determining two orthogonal axis and calculating tumor weight with the formula (/6)xa²xb, where a is the shorter and b is the longer axis. Lung metastases were counted by carefully examining the surface of the lungs, immediately after killing of the animals by cervical dislocation. Lungs were dissected into the five lobes, washed with PBS, and examined under a low-power microscope equipped with a calibrated grid. The weight of each metastasis was calculated by applying the same formula used for primary tumors and the sum of each individual weight gives the total weight of the metastatic tumor per animal.

Animal Studies

Animal studies were carried out according to the guidelines in force in Italy (DDL 116 of 21/2/1992) and in compliance to the Guide for the Care and Use of Laboratory Animals, Department of Health and Human Services Publication No. (NIH)86-23, Bethesda, MD, National Institutes of Health, 1985.

Tumor Lines for In Vitro Test

The cells used for in vitro test were three human-derived tumor lines: KB, MCF-7, and LoVo and TS/A murine adenocarcinoma. The established KB cell line (ECACC 86103004) was cultured according to standard procedures (Craciunescu et al., 1987). Vials of the original cell line were maintained in liquid N₂. The KB cell line was maintained in Eagle's minimum essential medium (Eagle, 1959) with 1% nonessential amino acids (Serva, Heidelberg, Germany), supplemented with 10% newborn calf serum (EuroClone, Devon, UK), and buffered with 3 mM tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid, 3 mM N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic acid, 3 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 3 mM tricine (Sigma Chemical Co., St. Louis, MO). Culture medium was added with penicillin-streptomycin solution (100 U/ml penicillin G and 100 µg/ml streptomycin; Sigma Chemical Co.). The established MCF-7 and LoVo cell line (ECACC no. 86012803 and no. 87060101) were cultured according to standard procedures (Soule et al., 1973; Drewinko et al., 1978) and maintained in Dulbecco's modified Eagle's medium/F-12 medium (Sigma Chemical Co.) supplemented with 10% FBS (Hyclone Europe, Holland), 2 mM L-glutamine (Hyclone Europe), and 100 U/ml penicillin G and 100 µg/ml streptomycin (Sigma Chemical Co.). TS/A adenocarcinoma cell line was kindly supplied by the group of Dr. G. Forni (Consiglio Nazionale delle Richerche, Centro di Immunogenetica ed Oncologia Sperimentale, Torino, Italy), was cultured according to standard procedures (Nanni et al., 1983) and was maintained in RPMI-1640 medium (Sigma Chemical Co.) supplemented with 10% FBS (Hyclone Europe), 2 mM L-glutamine (Hyclone Europe), and 50 µg/ml gentamycin sulfate solution (Irvine Scientific, Santa Ana, CA).

Cells from confluent monolayers were removed from flasks by 0.25% trypsin solution (Sigma Chemical Co.). Cell viability was determined by the Trypan blue dye exclusion test.

MTT Test to Evaluate In Vitro Cytotoxicity

Cell growth was determined by MTT viability test (Mosmann, 1983). Briefly, KB cells (5,000/well) were sown in 96-well cell culture clusters (Corning Costar, Milan, Italy) in culture medium and grown for 96 h; MCF-7, LoVo, and TS/A cells (50,000/well) were sown in 96-well cell culture clusters and grown for 24 h. Test compounds were dissolved in PBS containing Ca²⁺ and Mg²⁺ immediately before use and diluted to 10⁴ M concentration. The choice of this concentration is due to a preliminary test that showed the complete inactivity of lower concentrations, and to the fact that this concentration mimics that obtained in vivo in most organs and tissues with the test compounds after the 6-day cycle of administration for antitumor activity. Cells were then incubated 1 h with the test compounds, at 37°C with 5% CO₂ and 100% relative humidity. At the end of incubation time, drug solutions were removed and replaced with complete medium. The cytotoxic effect was evaluated 24 h after drug challenge: 10 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/ml in PBS, was added to each well of the 96-well culture plate containing 100 µl of medium and incubated for 4 h at 37°C with 5% CO₂ and 100% relative humidity. At this time the medium was discarded and 100 µl of acidified isopropanol (0.2 ml of 0.04 N HCl in 10 ml of isopropanol) was added to each well according to the modification used by Galeano et al. (1992). Optical density was measured at 540 nm on a spectrophotometer Multiskan MCC/340 (Labsystems OY, Helsinki, Finland).

Invasion Assay

Invasive ability was measured in a Transwell cell culture chamber (Costar) according to the method of Albini (1998). In brief, the lower surface of a polyvinylpirrolidone-free polycarbonate filter (24-mm diameter and 8-µm pore size) was coated with 200 µg/600 µl of Matrigel (Beckton Dickinson, Bedford, MA) and air dried overnight at room temperature. The filters were reconstituted with RPMI-1640 medium immediately before use. TS/A adenocarcinoma cell line, pretreated for 1 h with the test compounds (10⁴ M in PBS containing Ca²⁺ and Mg²) was treated with trypsine, collected by centrifugation, resuspended in RPMI-1640 supplemented with 10% FBS, and sown in triplicate in the upper compartment chamber (3.0 × 10⁵ cells/900 µl). The lower compartment was filled with RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and 50 µg/ml Gentamicin sulfate solution. Invasion was scored after 72 h and 96 h of incubation in a humidified CO₂ incubator at 37°C. After incubation, the filters were fixed with methanol (20°C) and stained with H&E. The cells on the upper surface of the filter were removed by a cotton swab. Tumor cells that had migrated from the upper to the lower side of the filter were counted by light microscopy at a magnification of 400×. The invasion was expressed as the percentage of total invasion compared with the original number of cells sown on day 0, calculated by the following formula:
For each experiment, the cells in at least six wells were counted.

Propidium Iodide Test

Viable cells (1 × 10⁶) of a single cell suspension, as determined by the Trypan blue exclusion test, were fixed in 70% ethanol at 4°C for at least 1 h. Before analysis the ethanol was removed by centrifugation and cells were washed twice with PBS. Cells were resuspended in PBS containing 1 mg/ml RNase at 37°C for 30 min and stained further for at least 30 min at room temperature in the dark with 40 µg/ml propidium iodide (Sigma Chemical Co.) (modified from Crissman and Steinkamp, 1973). Red fluorescence (610 nm) was analyzed using peak fluorescence gate to discriminate aggregates. Each analysis consisted of 10,000 events. Flow cytometry analyses were done at Fondazione Callerio with an EPICS XL flow cytometer. Cell cycle distribution was determined by analysis of data with Multicycle software (Phoenix Flow Systems, San Diego, CA).

Measurement of Ruthenium

Small pieces of primary tumor (about 0.3 g), an aliquot of about 0.4 ml of blood, 10⁶ cells of single cell suspensions, or the whole organ (liver, kidneys, lungs) were carefully weighed and frozen in Nalgene cryovials at 80°C.

Blood Analysis. Blood was left to melt at room temperature under gentle agitation to avoid foam formation. Then 100 µl of blood was put in another cryovial and treated with 25% tetramethylammoniumhydroxide (TMAH; Aldrich, Milan, Italy) in water, with a 1:5 sample:TMAH ratio, to completely digest the sample at room temperature in a closed vial, according to a procedure adapted from that described by Tamura et al. (1992).

Solid Tissue Analysis. A fragment of each organ, after careful weighing, was put in another cryovial and heated at 105°C until the fragment was completely dried. Weights were taken continuously and we considered that the specimen was completely dried when no further weight changes occurred. The fragment was then completely solubilized in the same cryovial (closed) by adding 0.5 ml of 25% TMAH in water at room temperature. After digestion, the volume was adjusted to 1 ml with Milli-Q water. Ruthenium was measured in triplicate by atomic absorption spectroscopy using a Varian SpectrAA-300 instrumentation, supplied with a graphite furnace mod GTA-96, an autosampler mod PSD-96, and a specific ruthenium emission lamp (Hollow cathode lamp Varian P/N 56-101447-00).

Statistical Analysis

Data were submitted to computer-assisted statistical analysis using the t test for grouped data, Dunnett's multiple comparison test, and Tukey-Kramer and Student-Newman-Keuls analysis of variance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In vitro challenge of human-derived (MCF-7, LoVo, and KB) and mouse (TS/A adenocarcinoma) cells with the test ruthenium compounds showed TEQU, but not NAMI-A, ICR, and trans-Ru, to significantly reduce tumor cell growth, as determined by the MTT test (Fig. 1). The reduction of cell growth caused by TEQU is statistically significant and independent of the cell type being treated, although the effect on MCF-7 cells is globally greater than that on the other cell lines.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   In vitro cytotoxicity of NAMI-A, ICR, TEQU, and trans-Ru on tumor cell lines. Data are expressed as percentage of the untreated controls. Bars represent the standard error of the mean values obtained from one experiment performed in triplicate. MCF-7, LoVo, KB, and TS/A cells, sown on 96-well cell culture clusters 24 h before (or 96 h for KB), were challenged with NAMI-A, TEQU, ICR, and trans-Ru for 1 h at 104 M concentration. MTT test was performed after 24 h of further cultivation in the appropriate medium. *P < .001 versus controls, ANOVA, Tukey-Kramer post test).

TS/A cell distribution among cell cycle phases, as detected by flow cytometry after propidium iodide staining of 1-h-exposed tumor cells, showed NAMI-A to increase the cells in G₂-M and to decrease the cells in G₀/G₁ phases. TEQU caused a marked reduction of cells in G₀/G₁, a correspondent increase of those in the S phase, and a certain amount of fragmented DNA, at values lower than those of G₀/G₁. ICR and trans-Ru caused no change compared with untreated controls (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effects of NAMI-A, ICR, TEQU, and trans-Ru on TS/A adenocarcinoma tumor cells distribution among cell cycle phases. Cells were treated as reported in Fig. 1; propidium iodide staining was performed after 24 h of further cultivation in the appropriate medium. Data for untreated controls are 50.4 ± 1.4 (%G0/G1), 38.6 ± 1.2 (%S), and 10.0 ± 0.9 (%G2-M), respectively. *P < .01 versus controls, ANOVA, Tukey-Kramer post test).

The study of ruthenium uptake by tumor cells, as determined by atomic absorption spectroscopy, showed TEQU significantly more concentrated in the treated cells than any other ruthenium complex (Fig. 3). Data of Fig. 3 lack the measurement of ruthenium uptake by MCF-7 cells treated with TEQU in that the number of tumor cells remaining after treatment and harvested from the plates was insufficient to perform the study. The comparison of ruthenium concentration in LoVo and TS/A cells treated with TEQU showed the former to uptake the compound at a 5-fold greater efficiency.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Ruthenium uptake by tumor cells in vitro challenged with NAMI-A, ICR, TEQU, and trans-Ru. Data are expressed as ruthenium content in 106 cells. Bars represent the standard error of the mean values obtained from one experiment performed in triplicate. MCF-7, LoVo, and TS/A cells, sown on six-well plastic plates 24 h before, were challenged with NAMI-A, TEQU, ICR, and trans-Ru for 1 h at 104 M concentration and immediately after harvested and processed for ruthenium determination by atomic absorption spectroscopy.

NAMI-A significantly inhibited tumor cell invasion of matrigel-coated membranes in a modified Boyden's chamber (Fig. 4). In the same experimental conditions, ICR and trans-Ru were completely inactive, whereas data on TEQU were omitted because all treated cells died and the few surviving remained on the upper side of the Matrigel membrane. An example of the appearance of the Matrigel-coated membranes invaded by the tumor cells is given by Fig. 5. In this figure, the TS/A cells treated with NAMI-A appear clustered on several strata on the upper side of Matrigel, whereas those treated with ICR and trans-Ru did not differ from those of untreated controls, and appeared infiltrated into the tubular structure of the Matrigel-coated membrane.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effects of NAMI-A, ICR, and trans-Ru upon invasion by TS/A adenocarcinoma tumor cells through matrigel-coated polycarbonate filters. Data represent cells that had completely traversed the matrigel-coated barrier after 72 and 96 h of incubation and are located in the lower compartment of the transwells. Data are expressed as percentage of the total cells originally added to the top well. Bars represent the standard error of the mean values obtained from one experiment performed in triplicate. **P < .001 versus controls, ANOVA, Tukey-Kramer post test.

View larger version (133K): [in this window] [in a new window]   Fig. 5.   Appearance of TS/A adenocarcinoma cells on the matrigel-coated polycarbonate filters 72 h after cell deposition. The filters were fixed with methanol, stained with H&E, and observed with a light microscope. The images are the upper surface of the filters. ECM, matrigel alone.

The effects of NAMI-A, TEQU, ICR, and trans-Ru on primary tumor growth and on lung metastasis formation in mice with advanced Lewis lung carcinoma are reported in Fig. 6. For this study we used each compound at its optimal dose level, a dose that gave the same host toxicity, considering the effects on body weight gain during treatment. In these conditions, TEQU and ICR significantly reduced primary tumor growth but were completely inactive on lung metastases. NAMI-A markedly inhibited metastasis number and weight, whereas trans-Ru was active only on metastasis weight. The analysis of ruthenium uptake by tumor cells of the primary tumor or by other tissues such as lungs, liver, kidneys, and whole blood in mice treated with the four test ruthenium compounds is reported in Fig. 7. The concentration of ruthenium found in the kidneys and in the primary tumor was similar, independent of the compound used. Conversely, the concentration of ruthenium after treatment with trans-Ru was significantly lower than that of NAMI-A and ICR in blood and in the lungs and was lower than that of NAMI-A, TEQU, and ICR in the liver. TEQU showed the lower level of ruthenium in the lungs of the treated mice. When we considered the overall amount of ruthenium found in each test organ, as a function of the total amount of ruthenium given to mice during the cycle of six administrations, TEQU appeared to be better retained by primary tumor, liver, and kidneys than the other three compounds, but it showed a rather poor uptake by lungs. Although NAMI-A and ICR showed a similar behavior, trans-Ru was the compound that showed the weakest propensity to accumulate in the test organs, including liver and kidneys.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effects of NAMI-A, ICR, TEQU, and trans-Ru on primary tumor growth and on lung metastasis formation in mice with Lewis lung carcinoma. Data are expressed as percentage inhibition of the controls. Bars represent the standard error of the mean values obtained from one experiment performed with groups of 10 BD2F1 mice, inoculated i.m. with 106 Lewis lung carcinoma cells on day 0, and treated i.p. with the indicated complexes on days 8 to 13. Primary tumor growth and lung metastases were determined on day 14 and 21 after tumor implantation, respectively. *P < .05, **P < .01 versus controls, Student-Newman-Keuls test.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Ruthenium uptake by host tissues and primary tumor in mice with Lewis lung carcinoma and treated with NAMI-A, ICR, TEQU, and trans-Ru. Three mice per group of the experiment of Fig. 2 were killed 24 h after last drugs administration and blood, liver, kidney, lung, and tumor were immediately harvested and processed for ruthenium determination by atomic absorption spectroscopy. Data are expressed as ruthenium concentration in each organ tested (ng/mg of wet tissue) (top) and as percentage of cumulative dose (bottom).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Ruthenium complexes were synthesized with the hope of reducing tumor cell growth by interacting with the most simple target of cancer chemotherapy drugs, i.e., DNA. Ruthenium complexes showed DNA interaction and suggested to achieve the selectivity of interacting only with DNA of cancer cells because of a selective uptake by tumor compared with healthy tissues (Sava et al., 1989) and because of a selective activation to cytotoxic species by the tumor tissue (Clarke et al., 1988). However, none of the ruthenium complexes tested to date have been more cytotoxic than cisplatin, the most appropriate reference control, based on another heavy metal of group VIII transition metals, although some of them showed moderate activity against platinum-resistant tumor cells (Coluccia et al., 1993, 1995). NAMI-A does not differ from this description, although it had a strange behavior in vivo: unlike cisplatin, which reduced primary tumor growth below 10% of controls but was inactive on host survival, it significantly increased the life-time expectancy of tumor-bearing mice without reducing primary tumor growth (Sava et al., 1999b). NAMI-A is now recognized as a novel antitumor agent endowed with interesting properties and with a mechanism of action comprising a consistent component of antiangiogenic properties, of inhibition of matrix metalloproteinase (Sava et al., 1996), and of modulation of tumor-tissue interactions (G. Sava, A. Bergamo, M. Magnarin, and M. E. Carotenuto, unpublished data), which places it among the new innovative agents for the treatment of metastases of solid tumors.

Because NAMI-A showed the capacity of controlling metastases of solid tumors it is interesting to have a model for screening other ruthenium complexes to find the most convenient for further development. In this search we gave particular importance to in vitro tests that may successfully address in vivo studies. In this context, the results of the present investigation seem to point out that a ruthenium compound, suitable for in vivo testing for antimetastasis activity, should in vitro 1) show no cytotoxicity for tumor cells; 2) induce the arrest of cells in the G₂-M premitotic phase, which however is transient and completely reversed by 48 h (Bergamo et al., 1999); and 3) inhibit matrigel invasion by tumor cells. In fact, of the four ruthenium complexes tested, only NAMI-A showed all these three properties in vitro and correspondingly it was very effective against metastasis in vivo. Certainly, the compound more cytotoxic in vitro, namely, TEQU, was devoid of antimetastasis activity in vivo. Apparently, the lack of activity of TEQU on lung metastases might be attributed to the poor propensity of the compound to bind to the lungs, provided that its cell uptake by tumor cells in vitro, on which it showed cytotoxic effects very closed to those already reported in other test systems, was enormously greater than that of the other compounds tested. However, ICR, whose binding to the lung was similar to that of NAMI-A, was totally inactive on metastases, whereas trans-Ru, which inhibited metastasis weight, was rather weakly bound to the lungs of the treated mice. Thus, the quality of in vitro activity is much more relevant than simple in vivo binding to the lungs for determining antimetastasis activity. This observation is relevant also for host toxicity, detected by the plasma levels of GPT and creatinine by common commercial kits, because, on liver and kidney, the high uptake of TEQU did not account for effects greater than those of trans-Ru whose uptake by these tissues was markedly lower (S. Zorzet, A. Sorc, and G. Sava, unpublished results).

Concerning NAMI-A and its in vitro inhibition of matrigel invasion by TS/A cells, it must be stressed that the histological appearance of the treated tumor cells on the matrigel-coated membrane accounts for the in vitro supposed increase of -integrin-mediated adhesion of cells to the extracellular substrate (R. Cramer and G. Zabucchi, unpublished data). Thus, these data further sustain the hypothesis that the effects of NAMI-A on lung metastases are mediated by the induced alterations on cell interactions with tissue matrix, which provides the essential signals to cell growth and invasion.

However, the main and more original conclusion of this article is that ruthenium complexes represent a basis for important anticancer drugs, endowed with an original activity, and that they can be screened by in vitro, low-invasive methods that may identify new agents, particularly active against solid tumor metastases. In particular, the in vitro study of a new compound, if providing the contemporary absence of direct cell cytotoxicity, associated to the capacity to inhibit matrigel crossing and a transient block of cells in the premitotic G₂-M phase, appears to be the prerequisite for a ruthenium compound to show in vivo selective antimetastasis effect, i.e., the capacity to inhibit the spontaneous metastases of solid experimental tumors. The exhibition of such characteristics by NAMI-A should also allow to state that the compounds that will show these properties would probably be associated to a reduced host toxicity in view of the mechanism of metastasis inhibition that avoids direct tumor cell killing. Although this model should not be proposed for compounds different from ruthenium compound yet, the validation of this test with other classes of compound should also allow an understanding of the combined weight of the reported phenomena for metastasis growth and control.