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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

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

Department of Biomedical Sciences (F.F., V.S., A.F., G.S.) and Department of Chemical Sciences (B.S., E.A.), University of Trieste; and Callerio Foundation Onlus (M.C., G.S.), Trieste, Italy

Received September 24, 2004; accepted December 2, 2004.

	Abstract

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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-imidazole dimethyl sulfoxide tetrachlororuthenate [(imH)[trans-RuCl₄(DMSO)(im)], NAMI-A, im = imidazole], is a ruthenium compound endowed in vitro with proadhesive effects (Bergamo et al., 2000; Sava et al., 2004) that has recently completed a phase I clinical trial at the Netherlands Cancer Institute (Rademaker-Lakhai et al., 2004). The effect on cell adhesion, as well as the capacity to inhibit angiogenesis and matrix metalloproteinases (Vacca et al., 2002), has been suggested to be responsible for in vivo NAMI-A activity on lung metastases of a number of solid tumors (Sava et al., 1998, 2003; Bergamo et al., 1999).

The evaluation of the effects of NAMI-A on cell adhesion (Sava et al., 2004) showed interesting connections with the complex multistep process of metastasis formation that involves a number of cell-cell and cell-matrix interactions before its completion. In particular, considering that in vivo NAMI-A pharmacokinetics show a rather slow release from the lungs (the site of experimental tumor metastases), it is important to know whether a prolonged in vitro exposure of tumor cells, even at low NAMI-A concentrations, might determine variations on the proadhesive effect observed after short treatments (Sava et al., 2004). With this study, we therefore evaluate the duration of the changes of KB tumor cell adhesion following exposure to NAMI-A and the influence of the compound secreted by the tumor cells previously exposed to a conventional short treatment. The possible role of integrin modulation in the NAMI-A proadhesive effect is also studied using fibronectin-coated plates and an integrin inhibitor, such as echistatin, a 49-amino acid peptide derived by Echis carinatus venom, a potent antagonist of IIb3, V3, and 51 integrins (Wierzbicka-Patynowski et al., 1999; Scheibler et al., 2001) that binds to the RGD integrin sequence (Thibault, 2000).

	Materials and Methods

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Compounds. NAMI-A was prepared by Dr. B. Serli. Fibronectin (FN) and echistatin were purchased from Sigma-Aldrich (St. Louis, MO).

Tumor Lines for in Vitro Testing. An established KB cell line, derived from a human epidermal carcinoma (European Collection of Cell Cultures number 86103004) was cultured as previously described (Frausin et al., 2002). The cell population doubling time was ca. 24 h. Cells from confluent monolayers were removed with 2 to 3 ml of trypsin solution (0.05% w/v) (Sigma-Aldrich) and subcultured once a week.

Cell Adhesion Assay. For studying NAMI-A cell proadhesive effects, KB cells were seeded at a density of 3 x 10⁴ cells/ml, in 0.2 ml/well in a 96-well plastic plate (Corning Costar, Milano, Italy) and incubated at 37°C as described previously (Sava et al., 2004). In experiments carried out on FN, each well was previously coated with 20 µg/ml FN. On the experimental day, nutritive medium was replaced by MEM with NAMI-A. At the end of the NAMI-A test, the incubation medium was replaced by fresh medium, and to assay NAMI-A proadhesive effect, at 24, 48, 72, and 96 h, plates were washed with PBS-calcium magnesium-free and 25 µl of MEM (control samples) or a trypsin solution (0.05% w/v) was added, and the plate was incubated at 37°C for 30 min. Adherent cells were quantified by the sulforhodamine B (SRB) assay (Sigma-Aldrich).

Sulforhodamine B Assay. To verify the entity of cell number in the plate wells, the adherent cells of each well were dyed with a colorimetric assay based on the quantification with sulforhodamine B of cellular proteic component (Skehan et al., 1990). Briefly, adherent cell cultures were fixed in situ by addition of 50 µl/well of cold 50% (v/v) trichloroacetic acid and were kept for 60 min at 4°C.

The supernatant was then discarded and the plates were washed two times with bi-distilled water and air-dried. SRB solution (0.4% w/v in 1% acetic acid) was added, and the cells were allowed to stain for 30 min at room temperature. Unbound SRB was removed by washing two times with 1% acetic acid. Then, the plates were air-dried. Bound stain was dissolved with unbuffered 10 mM Tris base [tris(hydroxymethyl)aminomethane] (Sigma-Aldrich), and the optical density was read at 570 nm with an automated microplate reader EL311s spectrophotometer (Bio-Tek Instruments, Winooski, VT). The modification in cell adhesion is expressed as adhesion increment percentage in samples incubated with NAMI-A, with respect to control samples. Each experiment was performed in sextuplicate and repeated twice.

Ruthenium Measurement by Flameless Atomic Absorption Spectroscopy. To evaluate intracellular ruthenium content, samples were processed according to a modification of the procedures by Tamura and Arai as described previously (Tamura and Arai, 1992; cited in Frausin et al., 2002); the amount of ruthenium released by KB cells in the extracellular medium was conversely read directly in the test samples. The concentration of ruthenium was measured in triplicate by means of graphite furnace atomic absorption spectrometry. The graphite furnace atomic absorption spectrometry instrument used was a Zeeman Graphite Tube Atomizer, model SpectrAA-300, supplied with a specific ruthenium emission lamp (Hollow cathode lamp P/N 56-101447-00) (Varian, Mulgrave, Victoria, Australia). To adjust for deterioration of the furnace during a daily work session, a reslope standard was done every six samples and accepted with less than 20% deviation. Changes in the readings of the reslope standard are included in the calculation of NAMI-A concentrations in the test samples. The lowest and highest limits of quantitation were set at concentration levels corresponding to the respective standard concentration. The limit of detection was estimated according to the EURACHEM guide. For the purpose of analytical methods, the fitness values of the lowest limit of quantitation, the highest limit of quantitation, and limit of detection, were, respectively, 12.5, 200, and about 8 ng Ru/ml sample. The quantification of ruthenium was carried out in 10-µl samples at 349.9 nm with an atomizing temperature of 2500°C, using argon as carrier gas at a flow rate of 3.0 l/min. Before each daily analysis session, a five-point calibration curve was obtained using Ruthenium Custom-Grade Standard, 998 mg/ml (Inorganic Ventures Inc., Lakewood, NJ).

In-Vitro-in-Vitro Bioassay. KB cells were seeded in a 96-well plastic plate (Corning Costar) and exposed at 0.1 mM NAMI-A as mentioned above. The 24-, 48-, or 72-h supernatants of NAMI-A-exposed cells, placed on untreated cells for 60 min, were removed, and plates were washed with PBS-calcium magnesium-free and processed for adhesion assay as described above.

NAMI-A Degradation in Physiological Saline. The evolution of NAMI-A during incubation in physiological saline-buffered solution at 37°C was monitored by electronic absorption spectroscopy in the visible range with a Jasco V-550 spectrometer (Jasco, Tokyo, Japan) equipped with a Peltier ETC-505T temperature controller.

Statistical Analysis. Data were analyzed using Student's t test. Significance was accepted with p < 0.05.

	Results

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NAMI-A Proadhesive Effect
Dependence of Time and Concentrations Test. The adhesion strength of KB cells, seeded on 96-well plastic plates and exposed to 0.001 mM NAMI-A (freshly prepared solutions) for different times (5, 10, 20, 30, and 60 min; Fig. 1A) or to 0.0001, 0.001, 0.01, and 0.1 mM NAMI-A for 10 or 60 min (Fig. 1B), was tested 24, 48, 72, and 96 h later.

View larger version (25K): [in this window] [in a new window]   Fig. 1. NAMI-A proadhesive effect: exposure time and concentration dependence. KB cells, seeded on 96-well plastic plates 5 days before, were exposed for different times to 0.001 mM NAMI-A (A) and for 10 min or 60 min to 0.0001 to 0.1 mM NAMI-A concentrations (B). Increase in adhesion strength was evaluated after 24, 48, 72, and 96 h of further cultivation, as described under Materials and Methods. Each value is the mean ± S.E. of two experiments, each performed in sextuplicate. , p < 0.05; , p < 0.01; , p < 0.001 compared with the previous value, Student unpaired t test.

At 24 h after 5-, 10-, and 20-min 0.01 mM NAMI-A exposure, cell adherence increased by about 10%, whereas after 30- and 60-min NAMI-A cell exposure, the observed increase of proadhesive effect was about 20% as compared with untreated cells (Fig. 1A). After 48 h, a further increase was appreciable at all NAMI-A exposure times. After 72 h, the percentage of increase was reduced in cultures exposed for the shortest, 5- and 10-min, NAMI-A treatments, whereas no difference in cell adherence compared with 48 h was observed in cultures exposed for 20, 30, and 60 min. After 96 h, the increase in cell adherence was reduced with all NAMI-A exposure times.

When cells were exposed to different NAMI-A concentrations (Fig. 1B), after 24 h, 0.0001 mM NAMI-A caused no modification of cell adherence, whereas higher concentrations increased it by about 15% independently of concentration and the length of exposure. After 48 h, cells exposed to NAMI-A for 10 min showed a concentration-dependent increase of cell adhesion, whereas cells exposed for 60 min did not show such dependence.

Influence of FN. The adhesion strength of KB cells seeded on FN and exposed for 60 min to 0.0001, 0.001, 0.01, and 0.1 mM NAMI-A was evaluated 24, 48, and 72 h later. A concentration-dependent increase of cell adherence appeared after 24 h and was reduced after 48 h only at the lowest concentration used (Fig. 2A). After 72 h, the same efficacy (about +20%) was maintained only in cells exposed to the highest (0.1 mM) NAMI-A concentration. The concomitant use of the integrin inhibitor echistatin completely abolished the proadhesive effect induced by fibronectin in control cells and that of low-dose NAMI-A; conversely, no significant change in the cell adhesion strength occurred in cells treated with higher concentrations of NAMI-A, indicating a dose-dependent competition on the same target for NAMI-A and echistatin and/or the possibility that other targets relevant for cell adhesion are involved at these doses (Fig. 2B).

View larger version (28K): [in this window] [in a new window]   Fig. 2. NAMI-A proadhesive effect: influence of fibronectin. KB cells, seeded on 96-well plastic plates which were previously coated with 20 µg/ml FN and grown for 5 days, were exposed for 60 min to 0.0001 to 0.1 mM NAMI-A concentrations. Increase in adhesion strength was evaluated after 24, 48, and 72 h of further cultivation, as described under Materials and Methods, without (A) or after treatment with echistatin (B). Each value is the mean ± S.E. of two experiments each performed in sextuplicate. , p < 0.05; , p < 0.01 compared with the previous value, Student unpaired t test.

Uptake and Release of NAMI-A by KB Cells
The intracellular and extracellular levels of ruthenium were measured after 5, 15, 30, 60, and 120 min and 24, 48, or 72 h after exposure of KB cells for 60 min to 0.1 mM NAMI-A. The amount of intracellular ruthenium at the end of the incubation time was 0.312 ± 0.027 µg/10⁶ cells (Fig. 3), corresponding to about 1% of that present in the 3-ml incubation medium. After 5 min, the amount of ruthenium released in the extracellular fluid was 0.310 ± 0.018 µg/10⁶ cells and did not vary in the next minutes. At 24 h after the end of cell exposure to NAMI-A, the extracellular levels of ruthenium were similar to the levels measured in the first minutes after the end of cell exposure and remained unaltered in the next days (data not reported). In general, the amounts of ruthenium remaining intracellularly were always ca. one-tenth of the total ruthenium present in the sample.

View larger version (12K): [in this window] [in a new window]   Fig. 3. NAMI-A uptake and release by KB cells. KB cells, seeded on six-well plastic plates and grown for 24 h, were exposed for 60 min to 0.1 mM NAMI-A; then, each well was washed twice and added with 3 ml of culture medium (MEM). After 5, 15, 30, 60, and 120 min cultivation samples of extracellular medium () and of cells () were processed as described under Materials and Methods. Each value is the mean ± S.E. of two experiments each performed in triplicate.

Proadhesive Effect of Ruthenium Released by KB Cells
The ability of the ruthenium species, released in the extracellular milieu by cells treated with NAMI-A, to increase cell adherence was tested on a fresh sample of KB cells challenged for 60 min with the culture medium (conditioned medium) obtained from 24-, 48-, or 72-h KB cells previously exposed to 0.1 mM NAMI-A for 60 min (Fig. 4). All the tested samples showed a pronounced capacity to increase cell adhesion: the adhesion strength increased along with the length of cell contact with the conditioned medium. These effects are completely reverted by the concomitant treatment with echistatin, indicating the involvement of adhesion molecules (1-integrins) in the ruthenium induction of cell adhesion.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Complete echistatin prevention of the proadhesive effects of cell-released NAMI-A. KB cells, seeded on 96-well plastic plates for 24 h and exposed to 0.1 mM NAMI-A for 60 min in MEM, were washed twice and added with 200 µl of the respective culture medium. After 24, 48, or 72 h at 37°C, the extracellular medium of this plate was put in contact for 60 min, with or without echistatin, with an untreated KB cell culture seeded on 96-well plastic plates 5 days before; any modifications of cell adherence were evaluated as described under Materials and Methods. Each value is the mean ± S.E. of two experiments each performed in sextuplicate. , p < 0.05; , p < 0.01 versus controls.

Proadhesive Effect of NAMI-A Solutions Aged in PBS or MEM
When the conditioned medium, obtained from 30 min of challenge of cells previously exposed to 0.1 mM NAMI-A for 1 h, was stored at 37°C and aged for 24, 48, and 72 h prior to evaluation of the proadhesive capacity, we obtained different results according to the medium used (Fig. 5). In fact, if KB cells were charged with NAMI-A in PBS and the conditioned medium was recovered in PBS, then a reduced ability of cells to adhere was measured. Conversely, when KB cells were treated in MEM and the conditioned medium was recovered in MEM, then a significant increase of cell adhesion was detected, comparable to those reported in Fig. 4, when aging of the conditioned medium occurred in the continuous presence of KB cells. By comparison, if NAMI-A was dissolved in PBS and aged for 24 to 72 h before challenge on KB cells, it completely lost its proadhesive properties. Rather, cells treated with such aged NAMI-A solutions appeared easier to detach by a diluted trypsin solution (Fig. 5). Conversely, when NAMI-A was dissolved in MEM and aged for the same times before cell challenge, a significant increase (p < 0.05) of cell adhesion occurred.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Dependence of NAMI-A proadhesive effects on cell treatment medium. KB cells, seeded on six-well plastic plates for 24 h and exposed to 0.1 mM NAMI-A for 60 min, respectively, in PBS or MEM, were washed twice and added with 3 ml of the respective culture medium. After 30 min of further cultivation, the extracellular medium of this plate (, PBS; or , MEM) was stored for 24, 48, or 72 h at 37°C in the dark prior to contact for 60 min with an untreated KB cell culture seeded on 96-well plastic plates 5 days before; any modifications of cell adherence were evaluated as described under Materials and Methods. For control, KB cells, seeded on 96-well plastic plates for 24 h, were exposed for 60 min to 0.1 mM NAMI-A previously stored, respectively, in PBS () or MEM (), for 24, 48, or 72 h at 37°C in the dark; any modifications of cell adherence were evaluated as described under Materials and Methods. Each value is the mean ± S.E. of two experiments each performed in sextuplicate. , p < 0.05; , p < 0.01; , p < 0.001 versus controls.

NAMI-A Degradation in Physiological Saline
In physiological saline solutions and at 37°C NAMI-A, [trans-RuCl₄(DMSO)(im)]^– undergoes stepwise hydrolysis of two chlorides within a few minutes in two well separated steps (as testified by the clean isosbestic points). The spectral changes in the visible region induced by chloride hydrolysis are shown in Fig. 6 for different NAMI-A concentrations; the left panels correspond to the hydrolysis of the first chloride, and the right panels to that of the second chloride. By monitoring the decrease in the absorption maximum characteristic of each species, it was possible to establish that the time to reach the maximum concentration of each of the hydrolyzed species RuCl₃(DMSO)(im)(H₂O) and [RuCl₂(DMSO)(im)(H₂O)₂]⁺ increased from 18 to 30 min and from 45 to 75 min, respectively, when NAMI-A concentration was reduced from 1 mM to 0.05 mM. Data of Fig. 7 show that [RuCl₂(DMSO)(im)(H₂O)₂]⁺ is significantly more active on cell adhesion than intact NAMI-A or its first neutral metabolite, RuCl₃(DMSO)(im)(H₂O).

View larger version (42K): [in this window] [in a new window]   Fig. 6. NAMI-A degradation in physiological saline. NAMI-A physiological saline solutions (0.05–1.0 mM) were monitored at 37°C by electronic absorption spectroscopy in the visible range. Left panels represent spectral changes during the first chloride hydrolysis; right panels represent spectral changes during the second chloride hydrolysis. Spectra were recorded every 2 min. Times needed to reach the maximum concentration of RuCl3(DMSO)(im)(H2O) (left panels) and [RuCl2(DMSO)(im)(H2O)2]+ (right panels) are indicated.

View larger version (24K): [in this window] [in a new window]   Fig. 7. Adhesive properties of NAMI-A-hydrolyzed chloride forms. NAMI-A physiological saline solutions (1 mM) aged at 37°C for 18 and 45 min, to obtain the maximum concentration of RuCl3(DMSO)(im)(H2O) first Cl– hydrolysis and [RuCl2(DMSO)(im)(H2O)2]+ second Cl– hydrolysis, respectively, were diluted to 0.1 mM. KB cells, seeded on 96-well plastic plates, were exposed for 10 min to these solutions. Any modifications of cell adherence were evaluated as described under Materials and Methods. Each value is the mean ± S.E. of two experiments each performed in sextuplicate. , p < 0.05; , p < 0.01 versus NAMI-A.

	Discussion

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The proadhesive effect of NAMI-A on KB cancer cells, which needs only 5 min of cell exposure to the rather low 0.001 mM concentration to appear, increases to a maximum at 48 h and lasts up to 72 h in the absence of further contact between cells and NAMI-A. This effect is even greater if NAMI-A treatment is prolonged from 5 to 10 min to 20 min. It is evident that the amount of ruthenium taken up by the cells regulates the effect on cell adhesion and that it is responsible for the duration of these effects, particularly considering that ruthenium species are rapidly released in the extracellular medium when NAMI-A is removed from the incubation medium. The prolonged action of NAMI-A could be important for its antimetastatic activity since the reduction of cell adherence can be considered one of the first steps in the metastatic progression, whereas the increase of cell adherence reduces cell ability to detach and to invade distant organs/tissues (Bashyam, 2002; Thompson and Price, 2002).

Concerning the mechanisms of activation of the proadhesive properties of tumor cells, we can suppose that a role is played by integrins. In fact, on KB cells seeded on FN, the peak of cell adhesion is anticipated to 24 h (instead of 48 h on plastic), and it is known how FN stimulates adhesion via integrin receptors (Kornberg et al., 1992; Fornaro et al., 2001). A further confirmation of integrin involvement is given by the complete prevention of NAMI-A's proadhesive effect (at low dosages) by the disintegrin molecule echistatin.

However, the process by which NAMI-A induces a proadhesive activity is more complex than the simple involvement of integrins in that high doses are not completely sensitive to the disintegrating effect of echistatin, suggesting the possibility for these doses of NAMI-A to interact with other targets, besides integrins, that have influence on cell cytoskeleton and which may influence cell adherence, such as the direct interaction with the polymerization of F-actin that leads to important changes of cell shape as observed by confocal microscopy in the same cells (Sava et al., 2004).

NAMI-A is a compound that undergoes a number of sequential transformations in PBS or other buffered saline solutions at pH 7.0 to 7.4 and 37°C, which include the rapid exchange of at least two chlorides with water molecules, and the partial hydrolysis of the axial DMSO group. The complex eventually evolves to polymeric oxo- or hydroxo-bridged species, characterized by dark color (Bouma et al., 2002; Sava et al., 2002; Alessio et al., 2004). This final process, however, is less likely to occur in vivo as it is considerably reduced in the presence of excess N-donor ligands (e.g., imidazole or peripheral histidine residues from serum albumin) which compete with OH^– for coordination to Ru(III) (Mestroni et al., 1994; Messori et al., 2000). Thus, it is to be expected that NAMI-A metabolites bind to some of the many bioligands found in cells and MEM buffer (e.g., amino acids, albumin, transferrin). The results that the ruthenium species extruded by KB cells increase the strength of cell adhesion, similarly to fresh NAMI-A solutions, add new and important knowledge to the pharmacological characteristics of NAMI-A. If we compare the inactivity of NAMI-A solutions aged in PBS and in MEM, we can suppose that the ruthenium species active on cell adhesion are NAMI-A metabolites bound to some component(s) of MEM buffer solution. As described above, the ruthenium species taken up by KB cells, upon incubation with NAMI-A, are almost immediately and completely released when the equilibrium with the extra cellular medium is removed. It is known that intracellular ruthenium is mainly distributed in cell nucleus besides cell membranes (Sava et al., 2004). The rapidity of cell extrusion of the ruthenium species suggests that they might be neutral.

The rapid cell release of NAMI-A metabolites can be reasonably thought to be responsible also for the low and reversible side effects on kidneys, the main way of ruthenium excretion and the organs in which its concentration is greater than in any other organ/tissue (Bergamo et al., 1999; Sava and Cocchietto, 2000; Cocchietto et al., 2003). This is confirmed also by the phase I study made at the Netherlands Cancer Institute in which renal toxicity, also at the toxic dose of 500 mg/m², was not relevant and was not considered responsible for the stop of dose escalation (Rademaker-Lakhai et al., 2004).

	Footnotes

 
Work was contributed by Ministero dell'Istruzione, dell'Università e della Ricerca and from the Metalli Anticancro Dell'Era postgenomica (MADE) project in Laboratorio per Identificare Nuovi Farmaci Antimetastasi. F.F. is a recipient of a fellowship grant from Fondazione C and D Callerio Onlus, Trieste.

doi:10.1124/jpet.104.078352.

ABBREVIATIONS: NAMI-A, imidazolium trans-imidazole dimethyl sulfoxide tetrachlororuthenate; FN, fibronectin; PBS, phosphate-buffered saline; MEM, minimum essential medium; SRB, sulforhodamine B.

Address correspondence to: Gianni Sava, Callerio Foundation Onlus, Via A. Fleming 22-31, 34127 Trieste, Italy. E-mail: g.sava{at}callerio.org

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