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CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY

Inhibition of B16 Melanoma Metastases with the Ruthenium Complex Imidazolium trans-Imidazoledimethylsulfoxide-tetrachlororuthenate and Down-Regulation of Tumor Cell Invasion

Department of Biomedical Science, University of Trieste (B.G., S.Z., G.S.), Trieste, Italy; Callerio Foundation-Onlus, Institute of Biological Research (M.C., G.S.), Trieste, Italy; and Reference Centre for Oncology (P.S.), Aviano, Italy

Received September 2, 2005; accepted December 15, 2005.

	Abstract

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The antimetastatic ruthenium complex imidazolium trans-imidazoledimethylsulfoxide-tetrachlorouthenate (NAMI-A) is tested in the B16 melanoma model in vitro and in vivo. Treatment of B6D2F1 mice carrying intra-footpad B16 melanoma with 35 mg/kg/day NAMI-A for 6 days reduces metastasis weight independently of whether NAMI-A is given before or after surgical removal of the primary tumor. Metastasis reduction is unrelated to NAMI-A concentration, which is 10-fold lower than on primary site (1 versus 0.1 mM), and is correlated to the reduction of plasma gelatinolitic activity and to the decrease of cells expressing CD44, CD54, and integrin-₃ adhesion molecules. Metastatic cells also show the reduction of the S-phase cells with accumulation in the G₀/G₁ phase. In vitro, on the highly metastatic B16F10 cell line, NAMI-A reduces cell Matrigel invasion and its ability to cross a layer of endothelial cells after short exposure (1 h) to 1 to 100 µM concentrations. In these conditions, NAMI-A reduces the gelatinase activity of tumor cells, and it also increases cell adhesion to poly-L-lysine and, in particular, to fibronectin, and this effect is associated to the increase of F-actin condensation. This work shows the selective effectiveness of NAMI-A on the metastatic melanoma and suggests that metastasis inhibition is due to the negative modulation of tumor cell invasion processes, a mechanism in which the reduction of the gelatinolitic activity of tumor cells plays a crucial role.

Ruthenium complexes represent a new class of compounds endowed with antitumor activity (Clarke, 1989; Keppler et al., 1989; Alessio et al., 2004a,b). NAMI-A, imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate, is one of these complexes, and it is characterized by a selective action against lung metastasis of solid mouse tumors and human xenografts (Sava et al., 1998, 2003; Bergamo et al., 1999). In vitro, NAMI-A inhibits tumor cell invasion of Matrigel-coated membranes in Transwell chambers (Zorzet et al., 2000; Sava et al., 2003) at doses free of cytotoxic activity on murine (TS/A) and human (MCF-7, LoVo, KB) tumor cell lines up to 0.1 mM (Bergamo et al., 1999; Zorzet et al., 2000; Sava et al., 2003). In vivo, NAMI-A selectively reduces metastasis formation. This is independent of whether it is given before surgery (early growing tumors) or after surgical ablation of primary tumor (already established metastases) (Zorzet et al., 2000; Sava et al., 2003). The postsurgical treatment of mice bearing MCa mammary carcinoma significantly improved the life span of the treated animals (Sava et al., 1999b).

The aim of this study was to investigate the in vitro and in vivo effects of NAMI-A treatment on murine melanoma cell lines. Murine melanoma cell lines differ from the previous models used in that they are relatively resistant to many cancer therapies (Sun and Schuchter, 2001). For the purpose of these experiments, we used the two variants of mouse melanoma B16F10 and B16 cell lines for in vitro and in vivo studies, respectively. In particular, we used the intra-footpad model, which mimics a subcutaneous growth of the tumor and allows removal of the primary tumor by surgery, approximately 10 to 15 days after implantation.

Because the adhesion process is involved in most, if not all, of the intermediate steps of the metastatic cascade (Honn and Tang, 1992), and adhesion of cells to extracellular matrix components is a prerequisite for cell movement to substrates, leading to migration and invasion, the capability of NAMI-A to interfere with tumor cell invasive activity was studied in vitro using a Transwell chamber assay. Furthermore, the effects of treatments on the expression of some important adhesion molecules involved in the metastatic spread such as CD44 (Birch et al., 1991), intercellular adhesion molecule-1 (Johnson, 1991), and integrin-₃ subunit (Seftor et al., 1992; Danen et al., 1994) were also investigated. Finally, we analyzed the effects of NAMI-A treatment on the gelatinases MMP-2 and MMP-9, enzymes whose expression in melanoma is associated with the conversion from radial growth phase to vertical growth phase and with subsequent metastasis formation (MacDougall et al., 1995). Because circulating metalloproteases could be considered as a prognostic marker for tumor malignancy, we also investigated the effects of treatments on the reduction of this protease activity in the serum of the treated tumor-bearing animals.

	Materials and Methods

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Compounds and Treatments. All reagents were purchased by Sigma (Milano, Italy) unless otherwise reported. NAMI-A was synthesized according to already reported procedures (Mestroni et al., 1998). For in vitro studies, a tumor cell line was incubated for 1 h with 1 to 100 µM NAMI-A in PBS Ca²⁺-Mg²⁺ saline solution, and analyses were performed 24 or 48 h the end of the treatment. In vivo treatment with NAMI-A was performed before or after the surgical removal of primary tumor. Presurgery treatment started when primary tumor had reached approximately 180 mg (range, 75–405 mg). NAMI-A was dissolved in isotonic nonpyrogenic physiological saline and was given intraperitoneum (i.p.) at the dose of 35 mg/kg for 6 consecutive days. Surgical removal of the primary tumor, proximal to the popliteal lymph node, was done 24 h after the end of the treatment; mice were anesthetized with Zoletil (70 mg/kg/200 µl i.p.) (Laboratories Virbac, Carros, France). Postsurgery treatment was done with the same dose and treatment schedule, starting 24 h after surgical removal of the primary tumor.

Tumor Cell Lines. B16F10 and B16 murine melanoma cell lines were used for in vitro and in vivo studies, respectively.

B16F10 cell line was obtained from the American Type Culture Collection (cat. no. CRL-6475; Manassas, VA) and maintained by twice-a-week passages in MEM (Euroclone, Wetherby, UK) supplemented with 10% fetal bovine serum (Euroclone), 1% of 10 U/ml penicillin and 100 µl /ml streptomycin, 2 mM L-glutamine, 100x nonessential amino acids, 1 mM sodium pyruvate (Euroclone), and 1 mM HEPES.

B16 melanoma cell line was obtained from the National Cancer Institute (Bethesda, MD) and maintained in vivo in C57BL/6 mice (Harlan, San Pietro al Natisone, Udine, Italy) by biweekly intramuscular (i.m.) implantation into the calf of the left hind leg. For experimental purposes, B6D2F1 female mice (Harlan) were injected intra-footpad with 0.5 x 10⁶/50 µl tumor cells of a single cell suspension, prepared from mincing with scissors the primary tumor mass obtained from donors implanted as previously described.

BAEC cell line was a kind gift from Dr. Paola Spessotto (Centro di Riferimento Oncologico, Aviano, Italy). BAEC cell line was maintained by weekly passage in Dulbecco's minimum essential medium (low glucose) (Euroclone) supplemented with 10% fetal bovine serum (Euroclone), 1% of 10 U/ml penicillin and 100 µl/ml streptomycin, and 2 mM L-glutamine (Euroclone).

Chemoinvasion Assay. The effect of NAMI-A on invasion activity of B16F10 tumor cells was assayed using a Transwell cell culture chamber (Corning Costar Italia, Milano, Italy). Briefly, polycarbonate filters with 8-µm pore size were precoated with 5 µl/50 µl fibronectin on the reverse side and dried at room temperature. Matrigel (5 µl/50 µl; Becton Dickinson Labware, Bedford, MA) was applied to the upper surface of the filter and dried overnight at room temperature.

Cells, sown 24 h after on multiwell plates, were treated with 1 to 100 µM NAMI-A in PBS Ca²⁺-Mg²⁺ for 1 h, and at the end of the treatment, cells were harvested with 1 mM EDTA in PBS and washed with serum-free MEM. Cell viability was determined by trypan blue exclusion dye test, and cells were resuspended to a final concentration of 1 x 10⁶/ml in MEM with 0.1% bovine serum albumin, 1% of 10 U/ml penicillin and 100 µl/ml streptomycin, and 2 mM L-glutamine (Euroclone).

One hundred microliters of cell suspension was added to the upper compartment and allowed to migrate for 24 h. The lower compartment was filled with conditioned medium from NIH-3T3 murine fibroblast cell line, supplemented with 10% fetal bovine serum. Cells remaining on the upper surface of the filter were removed by wiping them with a cotton swab. Cells on the lower surface were fixed with ice-cold methanol and stained with May-Grünwald-Giemsa. Seven to ten fields per filter were counted under a microscope at a magnification of 32x.

Trans-Endothelial Migration Assay. The effect of NAMI-A on trans-endothelial migration of B16F10 tumor cells was assayed using modified Transwell cell culture chamber. Briefly, polycarbonate filters with 8-µm pore size were precoated with 10 µl/50 µl Matrigel on the upper surface of the filter and dried overnight at room temperature. BAEC endothelial cells (0.8 x 10⁵/100 µl) were sown on Matrigel-coated filters in supplemented with 10% fetal bovine serum, 1% of 10 U/ml penicillin and 100 µl/ml streptomycin, and 2 mM L-glutamine. The lower compartments of the chambers were filled with complete medium. B16F10 tumor cells, sown 24 h after on multiwell plates, were treated with 1 to 100 µM NAMI-A in PBS Ca²⁺-Mg²⁺ for 1 h, and at the end of the treatment, cells were harvested with 1 mM EDTA in PBS and washed with serum-free MEM. B16F10 cells were labeled with the 5 mM FAST DiI probe (Molecular Probes, Eugene, OR) for 20 min at 37°C and then washed twice with PBS. Tumor cells were sown as previously reported, and they were allowed to migrate for 48 h. Cells remaining on the upper surface of the filter were removed, and cells on the lower surface were analyzed by fluorometric determination on FluoroCount-Packard reader (Packard, Meriden, CT).

Primary Tumor Growth and Lung Metastasis Evaluation. Primary tumor was determined by measuring two orthogonal axes with a caliper, and tumor weight was determined as:

(1)

where a and b are the shortest and the longest axis, respectively. Lung metastasis evaluation was performed at sacrifice. The number and size of metastasis were determined by means of a dissection microscope. Metastasis weight was calculated from their sizes applying the above formula (eq. 1), and the sum of each individual weight gave the total weight of the metastatic tumor per animal.

The effects of the treatment were represented by:

(2)

Analysis of Adhesion Molecules Expression on in Vitro Treated Cells. Flow cytometer analysis for CD44 and integrin-₃ receptor expression was performed 24 and 48 h after the end of the treatment. Cells were harvested with 1 mM EDTA in PBS, counted using trypan blue exclusion dye test, and 0.5 x 10⁶ viable cells were incubated for 1 h at 4°C with rat monoclonal antibody against murine CD44-FITC-conjugate (Southern Biotechnology Associates Inc., Birmingham, AL) and hamster monoclonal antibody against integrin-₃-phycoerytrin-conjugate (Santa Cruz Biotechnology, Santa Cruz, CA). Nonspecific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies (Southern Biotechnology Associates Inc.). At the end of incubation, cells were washed twice with PBS containing 0.5% bovine serum albumin and 0.1% NaN₃ and resuspended in 1% paraformaldehyde in PBS.

Analysis of Adhesion Molecule Expression and of Cell Cycle Distribution on in Vivo Treated Tumor Cells from Primary Tumor and from Lung Metastases. Flow cytometer analysis of cell cycle phase distribution and CD44, CD54, and integrin-₃ receptor expression were performed on primary tumor cells at surgery and on metastatic cells isolated from lungs at sacrifice. Cells from the primary tumor mass or from the metastatic nodules were mechanically disaggregated and filtered through a double layer of sterile gauze. For the receptor analyses, cells were incubated for 1 h at 4°C with rat monoclonal antibody against murine CD44-FITC conjugate (Southern Biotechnology Associates Inc.), rat monoclonal antibody against murine CD54-FITC conjugate (Southern Biotechnology Associates Inc.), and hamster monoclonal antibody against integrin-₃-phycoerytrin conjugate (Santa Cruz Biotechnology). Isotype controls were analyzed in parallel. Then cells were fixed with 1% paraformaldehyde for 15 min at 4°C and washed with ice-cold PBS. Cells were permeabilized with 100% ice-cold methanol for 15 min, washed, and resuspended in 0.1% saponin in PBS containing 2% fetal bovine serum for 30 min at room temperature. After washing, cells were resuspended in 1 ml of PBS containing 0.5% bovine serum albumin and 0.1% NaN₃ and analyzed on flow cytometer.

For the cell cycle analysis, 0.5 x 10⁶ cells of a single cell suspension of B16 melanoma cells, obtained from the primary tumor mass or from lung metastases and prepared as described above, were fixed in 70% ethanol at 4°C for at least 1 h. Ethanol was removed by centrifugation, and cells were washed twice with PBS; cells were resuspended in PBS containing 1 mg/ml ribonuclease type A, kept at 37°C for 30 min, and further stained with propidium iodide (40 µg/ml) for at least 30 min at room temperature in dark. Red fluorescence (excitation at 488 nm and emission at 610 nm) was analyzed using peak fluorescence gate to discriminate aggregates.

Flow Cytometry Analysis. Flow cytometry analyses were done with a CYTOMIX FC500 flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Each analysis consisted of 10,000 events. Cell cycle distribution was determined by analysis of data with Multicycle software (Phoenix Flow Systems, San Diego, CA). Antibody analysis was performed by WinMDI 2.8 software (Dr. J. Trotter, Scripps Research Institute, La Jolla, CA).

Adhesion Assay (Resistance to Trypsin Treatment) on B16F10 Cells. Cell adhesion assay was performed on different substrates on 96-well plates (Falcon, Franklin Lakes, NJ), using fibronectin from human plasma and poly-L-lysine (70–150 kDa). Wells were coated with 50 µl of fibronectin (the original product was diluted to 20 µg/ml with sterile apyrogenic water for injection; Laboratori Diaco Spa, Trieste, Italy) or with 100 µl of a 10 µg/ml solution poly-L-lysine. Plates were kept in a humidified cell-culture chamber at 37°C for 4 h, and each plate was washed twice with sterile PBS before cell sowing. Briefly, 1 x 10⁴ B16F10 cells in 200 µl of complete medium were sown and incubated at 37°C. After 2 days, the medium was removed, and cells were washed with PBS and added with complete medium, with or without 100 µM NAMI-A. After 1 h exposure, the supernatant was discarded, and plates were washed twice with PBS; then 25 µl of a (0.05% w/v) trypsin solution was added to each well, and plates were kept at 37°C for 30 min. Control wells were run without the trypsin solution. After incubation, trypsin was removed, wells were washed with PBS, and nondetached cells were fixed with 200 µl of a solution (10% w/v) of ice-cold trichloracetic acid for 1 h at 4°C. After fixation, trichloroacetic acid was removed and wells washed twice with distilled water. Plates were then left to dry at room temperature for few minutes before determining the number of fixed cells by the sulforhodamine B test.

Sulforhodamine B Assay. Adherent cells in each well were stained with a colorimetric assay based on the quantification with sulforhodamine B of the cell protein component (Skehan et al., 1990). Briefly, 50 µl of a sulforhodamine solution [0.4% (w/v) in 1% acetic acid] was added to each well, treated as described above, and cells were allowed to stain for 30 min at room temperature. Unbound sulforhodamine was removed by washing twice with 1% (v/v) acetic acid. Plates were air-dried, the bound stain was dissolved with unbuffered 10 mM Tris base (Tris-hydroxymethylaminomethane, pH 10.5), and the optical density was read at 570 nm with an automatic computerized spectrophotometer (SpectraCount; Packard).

The modification of cell adhesion is expressed as the percentage of adhesion increment in the samples treated with NAMI-A compared with control samples and with similar samples incubated without trypsin solution. Each experiment was performed with 14 replicates and repeated twice.

Confocal Microscopy Visualization of Actin Filaments. The great affinity of phalloidin, a poisonous alkaloid obtained from Amanita phalloides, for actin is useful for visualizing actin filaments (F-actin) inside cells (Herzog et al., 2004). Approximately 10⁴ B16F10 cells were sown in 400 µl of complete medium on an eight-well chamber slide (FALCON, Culture Slide; Becton Dickinson, Franklin Lakes, NJ) and incubated for 72 h at 37°C with 5% CO₂. Cells were then treated with PBS containing 100 µM NAMI-A for 1 h at 37°C. After this challenge, cells were washed twice with PBS Ca²⁺-Mg²⁺ and fixed for 15 min at room temperature with 200 µl of a 4% (w/v) solution of paraformaldehyde prepared in PBS. After two washings with PBS Ca²⁺-Mg²⁺, cells were permeabilized for 2 min at room temperature with a solution of PBS with 0.1% (w/v) bovine serum albumin containing 0.1% (v/v) Triton X-100 (all from Sigma) and 4% (v/v) of fetal bovine serum (HyClone, Logan, UT). Then, after two washings with PBS with 0.1% bovine serum albumin, cells were incubated for 1 h in the dark at room temperature with 100 µl of 4 U/ml (in PBS) phalloidin (Alexa Fluor 488 phalloidin; Molecular Probes). Finally, chambers were removed from the slides, covered with Mowiol (a substance that shelters the fluorescence; Calbiochem San Diego, CA) and with a cover slide. B16F10 cells were then observed with a microscope (DiaPHOT-200; Nikon, Tokyo, Japan), supported with a confocal system (MCR-1024; Bio-Rad Laboratories, Hercules, CA). The "pseudo-colorized" image elaboration was done with the LaserSharp Processing associated software (Bio-Rad Laboratories).

Zymography Assay. In in vitro studies, serum-free supernatants of B16F10 cells incubated for 1 h with 1 to 100 µM NAMI-A were collected 24 h after the end of the treatment and were concentrated 20x using Centricon Y-10 tubes (Millipore, Bedford, MA). Equal volume of concentrated medium was used for the detection of gelatinase activity.

In in vivo studies, gelatinolitic activity was evaluated on plasma fraction obtained from blood taken by cardiac puncture from anesthetized animals at sacrifice. Mice were anesthetized with sublethal doses of ethylurethan (1.5 g/kg i.p.) (Carlo Erba, Milano, Italy). To avoid blood coagulation, 50 µl of 0.1 M sodium citrate was used. Plasma was obtained by centrifugation at 10,000g for 20 min at room temperature and was aliquoted and frozen at –80°C until processing. Euglobulin fraction was prepared by mixing 0.1 ml of plasma with 0.9 ml of deionized ice-cold water, acidified to pH 5.5 with 40% (v/v) acetic acid. This mixture was incubated for 1 h at 0°C and centrifuged for 10 min at 5000 rpm. Euglobulin was then dissolved in 0.1 ml of PBS, pH 7.4 (modified from Ranuncolo et al., 2000). Twenty-one microliters of euglobulin fraction were mixed with electrophoresis sample buffer.

All samples were electrophoresed in 10% polyacrylamide-separating gel in the presence of 1% SDS and containing 1 mg/ml gelatin, without reducing or heating. Then gels were washed twice for 30 min with 2.5% Triton X-100 to remove SDS and finally washed three times for 5 min with distilled water. Gels were incubated overnight with collagenase buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 5 mM CaCl₂). They were stained for 4 h in 30% methanol/10% glacial acetic acid containing 0.1% Coomassie Brilliant Blue R-250 and destained with the same solution without dye. Gelatinolitic activity was detected as clear bands on the blue background. Molecular weight standard (6500–205,000) was run on each gel.

Atomic Absorption Spectroscopy Measurement. Ruthenium uptake from primary tumor cell and lungs was analyzed by atomic absorption spectroscopy. Briefly, 1.0 x 10⁶ cell from primary tumor cell suspension obtained at surgery or fragment of lungs obtained at sacrifice was dried overnight at 80°C and then at 105°C in Nalgene cryovials (Nalge Company, Rochester, NY). Cell decomposition was facilitated by the addition of tetramethylammoniumhydroxyde (25% in water) (Aldrich Chimica, Milano, Italy) and Milli-Q water at a ratio of 1:1 directly in each vial at room temperature and under shaking (modified from Tamura and Arai, 1992). Final volumes were adjusted to 1 ml with Milli-Q water. Ruthenium concentration was measured using a graphite furnace atomic absorption spectrometer, model SpectrAA-220Z, supplied with GTA 110Z power and a specific ruthenium emission lamp (hollow cathode lamp P/N 56-101447-00; Varian, Mulgrave, VIC, Australia). The lower and higher limits of quantification were set at the levels corresponding to the lower (20 ng of ruthenium per milliliter) and higher (100 ng of ruthenium per milliliter) standard concentrations, respectively. The limit of detection 10 ng of ruthenium per milliliter was estimated according to the EURACHEM guide (http://www.eurachem.ul.pt/guides/valid.pdf, 1998). The quantification of ruthenium was carried out in 10-µl samples at 349.9 nm with an atomizing temperature at 2500°C, using argon as the 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 µg/ml (Inorganic Ventures Inc., Lakewood, NJ).

Animal Studies. Animal studies were carried out according to the guidelines enforced in Italy (DDL 116 of 21/2/1992 and subsequent addenda) and in compliance with the Guide for the Care and Use of Laboratory Animals, Department of Health and Human Services publication no. 86-23 (National Institutes of Health, Bethesda, MD, 1985).

Statistical Analysis. Experimental data were subjected to computer-assisted statistical analysis using analysis of variance and the Student-Newman-Keuls to make comparisons within and between groups to compare the treated groups versus the untreated controls. Differences of p < 0.05 were considered to be significantly different from controls.

	Results

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In Vitro Results. NAMI-A was virtually inactive on B16-F10 melanoma tumor cell viability up to 0.1 mM concentration and 96-h cell exposure, as evaluated by the trypan blue exclusion test (data not shown). In the same experimental conditions, NAMI-A statistically reduced B16F10 tumor cell invasion, as detected with a Transwell chamber in a chemoinvasion assay (Fig. 1, bars on the left). Twenty-four hours after cell sowing, 1 µM NAMI-A inhibited tumor cell invasion by 50.1 ± 5.8% (p < 0.001 versus controls), whereas 100 µM NAMI-A reduced invasion up to 85.8 ± 3.1% (p < 0.001 versus controls). Similarly, NAMI-A inhibited tumor cell invasion when the treated cells were sown on a layer of endothelial cells (Fig. 1, bars on the right), although in this case the reduction of invasion is less pronounced, being approximately 50% at the maximum tested dose. The morphology of the cells, as they appeared after invasion on the lower surface of the filter, showed flattened and rounded cells with a reduction of pseudopodia formation compared with untreated controls. In particular, 100 µM NAMI-A-treated cells looked well rounded and free of pseudopodia formations (Fig. 2).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Effects of NAMI-A on extracellular matrix invasion and on trans-endothelial migration of B16F10 cells. On the extracellular matrix invasion assay, 1 x 105/100-µl cells pretreated with 1 to 100 µM NAMI-A were sown on the top compartment of a Transwell chamber with polycarbonate filters of 8-µm pore size, precoated with 5 µl/50 µl fibronectin on the reverse side and with 5 µl/50 µl Matrigel onto the upper surface. Invading cells per field on the lower surface were counted in seven predetermined fields 24 h later. On the trans-endothelial migration assay, 0.1 x 105 B16F10 tumor cells, pretreated with 1 to 100 µM NAMI-A and labeled with FAST DiI fluorescent probe, were sown on Transwell chamber filters coated with 10 µg/ml Matrigel and a monolayer of endothelial cells. Tumor cells were allowed to invade for 48 h at 37°C. Invading cells were analyzed as reported under Materials and Methods. Each value is the average of triplicate samples and is expressed as a percentage of inhibition of invasion versus controls. Statistics: Student-Newman-Keuls test (**, p < 0.01; ***, p < 0.001 versus controls).

View larger version (59K): [in this window] [in a new window]   Fig. 2. Light microscopic morphology of the invading B16F10 cells. Representative images of B16F10 melanoma cells adherent to the lower surface of the Transwell filters after invasive process. Cells were stained with May-Grünwald-Giemsa dye, and images were acquired under a light microscope at a magnification of 32x.

Similarly, using a modified Transwell chamber assay adapted to study trans-endothelial migration 48 h after cell sowing, NAMI-A reduced B16F10 migration throughout the endothelial layer at any dose tested. The higher activity was observed at 100 µM; at this dose, tumor cell migration was inhibited by 48.6 ± 3.5% (p < 0.001 versus controls) (Fig. 1, bars on the right). Invasion inhibition is associated to a dose-dependent reduction in gelatinase activity of the treated cells (Fig. 3, left). MMP-2 activity, measured in the supernatant of the treated cells, significantly decreased at 100 µM NAMI-A, whereas MMP-9 activity, which was basically lower than MMP-2, was not modified by any treatment.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Analysis of the gelatinolitic activity on the serum-free conditioned medium of in vitro-cultured B16F10 melanoma cells or on the plasma fraction of mice with B16 melanoma and treated with NAMI-A. Serum-free supernatants of B16F10 melanoma cells, exposed for 1 h to 1 to 100 µM NAMI-A, were collected 24 h after the end of the treatment and concentrated using Centricon Y-10 tubes. Plasma euglobulin fractions were obtained at sacrifice from blood samples, taken by cardiac puncture in open-chest anesthetized mice carrying intrafood B16 melanoma implants. Mice were treated i.p. with 35 mg/kg/day NAMI-A for 6 consecutive days, on days 15 to 20 (before surgery) or 22 to 27 (after surgery). Gelatinolitic activity was detected as clear bands on the blue background after Coomassie staining. none, controls; early, NAMI-A before surgery; late, NAMI-A after surgery.

View this table: [in this window] [in a new window]   TABLE 1 CD44 and integrin-3 expression on B16F10 melanoma cells in vitro after exposure to NAMI-A B16F10 melanoma cells (0.5 x 106), exposed for 1 h to 100 µM NAMI-A, were labeled 24 and 48 h after the end of treatment with monoclonal antibodies rat anti-mouse-CD44 and hamster anti-mouse integrin-3 (2 µg/10 µl) and processed by flow cytometry. At least 10,000 events were acquired for each sample. Flow cytometry data were processed by computational analysis using WinMDI software and are the mean ± S.E. of sextuplicate samples obtained from two separate experiments. No statistical differences between controls and NAMI-A-treated cells; Student-Newman-Keuls test.

View larger version (109K): [in this window] [in a new window]   Fig. 4. Confocal microscopic images of B16F10 melanoma cells exposed in vitro to NAMI-A. F-actin distribution is revealed at the confocal microscope by fluorescent phalloidin. Original magnification, x100. More details are presented under Materials and Methods. Left, control untreated cells sown on fibronectin or on poly-L-lysine; right, cells sown on fibronectin or on poly-L-lysine and treated with 100 µM NAMI-A for 1 h.

In Vivo Results. In vivo NAMI-A treatment was devoid of significant effects on primary tumor growth at the dose and treatment schedule used and at all of the times analyzed (unreported results). Interestingly, NAMI-A significantly reduced metastasis weight either when given before surgical removal of the primary tumor (early phases of metastasis formation) or after surgery (34.5 ± 7.3 and 35.0 ± 7.4%, respectively, for pre- and postsurgical treatments; p < 0.05 versus controls); no similar effect was observed on metastasis number (Fig. 5). Considering the distribution of tumor cells among cell cycle phases, NAMI-A was free of effects at primary tumor level, although it significantly increased the percentage of cells in G₁ phase and decreased the percentage of those in S phase in lung metastasis cells (p < 0.05 versus controls) (Fig. 6). The study of the expression of CD44, CD54, and integrin-₃ on single cell suspensions was performed on primary tumor (prepared 24 h after last dosing) and on lung metastases (harvested at sacrifice for metastasis evaluation). We showed no modification of the density of CD44 and CD54 receptors per cell or of the percentage of positive cells on primary tumor cells. Conversely, NAMI-A significantly reduced the density of integrin-₃ receptor per cell unit (p < 0.05 versus controls). However, the expression of these adhesion molecules was significantly affected by NAMI-A on the metastatic cells with a statistically significant decrease of the percentage of positive cells (Table 2). It must also be stressed that the percentage of CD44 and integrin-₃ positive cells in the treated samples was lower than that found in vitro on cultured cells. The difference was greater for primary tumor cells than for cells harvested from lung metastases.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Primary tumor growth and lung metastasis formation in mice with B16 melanoma and treated with NAMI-A. Data are expressed as mean ± S.E. and represent the pool of results obtained from three separate experiments performed in the same conditions. In the first experiment, NAMI-A was given on days 12 to 17 (before surgery); in the second experiment, NAMI-A was given on days 10 to 15 (before surgery); in the third experiment, NAMI-A was given on days 15 to 20 (after surgery). B6D2F1 female mice were implanted intra-footpad with 0.5 x 106 B16 melanoma cells on day 0. Primary tumor growth was measured at surgery (day 18, day 16, and day 21, respectively, for the first, second, and third experiment), and lung metastases were evaluated at sacrifice on days 34, 36, and 37, respectively, for the first, second, and third experiment. Statistics: Student-Newman-Keuls test (*, p < 0.05 versus controls).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Cell distribution among cell cycle phases of B16 melanoma cells harvested from primary tumor and from lung metastasis. Single cell suspension was obtained from primary tumor mass or from lung metastatic nodules of the same mice treated presurgery with 35 mg/kg/day NAMI-A for 6 consecutive days. Tumor (0.5 x 106) or metastatic (0.25 x 106) cells were labeled with propidium iodide solution as described under Materials and Methods. Cell cycle distribution was determined by analysis of data with Multicycle software. Data are expressed as percentage of cells on cell cycle phases and were obtained from two experiments and are the mean ± S.E.M of five different samples. Primary tumor growth was measured at surgery (day 18 and day 16, respectively, for the first and the second experiment), and lung metastases were evaluated at sacrifice on days 34 and 36, respectively, for the first and the second experiment. Statistics: Student-Newman-Keuls test (*, p < 0.05 versus controls).

View this table: [in this window] [in a new window]   TABLE 2 CD44, integrin-3 and CD54 expression on B16 melanoma cells harvested from the primary and metastatic lesions B16 melanoma cells (0.5 x 106) obtained from primary tumor cell suspensions at surgery and 0.25 x 106 B16 melanoma cells obtained from metastatic lung nodules at sacrifice were labeled with monoclonal antibodies rat anti-mouse-CD44, rat anti-mouse-CD54, and hamster anti-mouse integrin-3 (2 µg/10 µl) and processed by flow cytometry. At least 10,000 events were acquired for each sample. Flow cytometry data were processed by computational analysis using WinMDI software and are the mean ± S.E. of five different samples (one sample per mouse). Data were obtained from two separate experiments. Primary tumor growth was measured at surgery (day 18 and day 16, respectively, for the first and second experiment), and lung metastases were evaluated at sacrifice on days 34 and 36, respectively, for the first and the second experiment. Statistics: Student-Newman-Keuls test.

In the treated animals, NAMI-A markedly reduced the level of circulating gelatinases (Fig. 3, right). In particular, the postsurgical treatment reduced both MMP-9 proform (92 kDa) and its active form (82 kDa) to a great extent; MMP-2 proform (72 kDa) and its active form (62 kDa) were similarly reduced, but the reduction was less pronounced.

The concentration of ruthenium in the primary tumor, measured in the treated mice 24 h after last dosing, was relatively high, being around 1 mM [in general, greater than that found in mice carrying i.m. MCa mammary carcinoma or Lewis lung carcinoma and dosed i.p. with the same schedule used in this study (0.74 ± 0.38, 0.74 ± 0.20 x 10^–4, respectively; Cocchietto et al., 2003)] (Table 3). Ruthenium concentration evaluated at sacrifice, in the lungs of the same animals, was still relatively high and, supposing it could represent NAMI-A in its active form, was of the same order of magnitude of that used for the in vitro studies.

	Discussion

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NAMI-A is the first anticancer drug based on ruthenium to enter clinical trials, having recently completed a phase 1 study at The Netherlands Cancer Institute. It showed a good tolerability, following a cycle of slow daily i.v. infusion for 5 days with a 3-week interval between cycles. A large proportion of the treated patients received more than one cycle and up to eight cycles (Rademaker-Lakhai et al., 2004). In preclinical studies, NAMI-A showed pronounced antimetastatic effects on animal tumors such as MCa mammary carcinoma (Sava et al., 1998, 1999a,b) and Lewis lung carcinoma (Zorzet et al., 2000; Cocchietto et al., 2003) or on human xenograft models such as H460M2 lung carcinoma (Sava et al., 2003) after i.p. treatments for 6 consecutive days. This was independent of whether dosing occurred at early or advanced stages of tumor growth.

The present study shows the effectiveness of NAMI-A on another tumor known as the B16 murine melanoma and suggests that metastasis inhibition is due to the negative modulation of tumor cell invasion processes. The modulation of invasion processes induced by NAMI-A is documented by the reduction of gelatinase activity, by the inhibition of cell crossing of Matrigel and of endothelial barriers, and by the marked changes on cell shape and F-actin-dependent cytoskeleton organization. In previous studies using HeLa tumor cells, NAMI-A induced the rearrangement of cytoskeleton, interfering with the F-actin polymerization/depolymerization process. This induced the formation of globular and fibrous cordons, leading to the increase of the adhesive strength (Sava et al., 2004). Previous studies have also described how NAMI-A increases the adhesion strength to the substrate on KB tumor cells and how the ignition of this effect likely depends on integrin activation (Frausin et al., 2005). The integrity of the cytoskeleton structure is necessary for the control of cell motility, because the alteration of its architecture modifies cell morphology and can induce the rearrangements of adhesion molecules, with modification of cell motility. Ballestrem et al. (2000) showed the involvement of F-actin polymerization and microtubule assembly in the motility and migration of B16 melanoma cell line. The study underlined the role of microtubules in the regulation of cellular translocation, migration, and the role of actin fibers in controlling protrusive lamella formation. Metalloprotease activation is also mediated by an intact cytoskeleton. Compounds that alter cytoskeleton structure may interfere with this process (Tomasek et al., 1997). In addition, the reduction of MMP release leads to the reduction of the tumor cell capability to degrade the extracellular matrix and to invade (Stearns and Wang, 1992; Westerlund et al., 1997).

All these events find their reinforcement in the modulation of adhesion molecules such as CD44 and integrin-₃. The expression of these receptors is particularly modified in lung metastases, the preferential target for the antitumor effects of NAMI-A.

In vivo metastasis inhibition by NAMI-A is not a new concept, having already been described in detail in previous studies (Sava et al., 1998, 2003; Bergamo et al., 1999). Similarly, the in vitro effects of this ruthenium-based compound on gelatinases, invasion, and F-actin organization have been described previously (Sava et al., 2004). However, this is the first time the effects of NAMI-A are described, in vitro and in vivo, on the same tumor cell line. According to these studies' results, it is possible to attribute the reduction of metastasis growth to a specific mechanism of modification of the tumor cell behavior.

NAMI-A reduces metastasis growth of B16 melanoma. This reduction has a greater effect on metastasis weight than on metastasis number, correlating with what has been observed for other carcinomas (MCa mammary carcinoma, Lewis lung carcinoma, and H460M2 lung carcinoma). Thus the effects of NAMI-A should not simply be attributed to the prevention of metastasis formation, but they imply a selective interference with the metastatic proliferation. Comparing the effects of NAMI-A treatment on the distribution of cells among cell cycle phases, we found that it significantly increased the percentage of metastatic cells in G₁ phase and decreased the percentage of metastatic cells in S phase. This induced a less proliferative phenotype. Similar effects were not observed on tumor cells at the primary site. This suggests that also in B16 melanoma, NAMI-A seems to be selectively effective on metastatic cells rather than on primary tumor cells. This result is further stressed by a statistically significant reduction of in vivo expression of CD44, CD54, and ₃ integrin molecules on metastasis rather than on primary tumor.

The integrin-₃ receptor has a number of important roles in the angiogenesis process, in binding active MMP-2 (Brooks et al., 1996; Deryugina et al., 1997) and also in trans-endothelial migration (Voura et al., 2001). An increase in integrin-₃ expression on melanoma cells correlates with an increase in in vivo proliferation (Li et al., 2001; Trikha et al., 2002), and there is an association with the transition from noninvasive radial growth phase to metastasizing vertical growth phase (Albelda et al., 1990). The reduced expression of this receptor in our conditions and the supposed role of integrin-₃ in the activation of the adhesion strength observed on KB cells (Frausin et al., 2005) stresses the possibility that the selective activity of NAMI-A on metastatic cells might depend, at least in part, on the interaction with integrin receptors leading to the inhibition of some important steps involved in the metastatic process. In addition, NAMI-A treatment also altered the expression of adhesion molecules on metastasis cells.

Ruthenium concentration (and therefore the in vivo available forms of NAMI-A) in the primary tumor mass of B16 melanoma is approximately two orders of magnitude higher than that found in other tumors (1.64 ± 0.23 versus 0.08 ± 0.01 mM) (Cocchietto et al., 2003), and this fact could be only in part ascribed to the particular site of implantation of this tumor (i.e., subcutaneously into the footpad instead of intramuscularly into the calf of the hind leg). It is known that in vivo, after repeated i.p. NAMI-A injections, ruthenium accumulates in the skin more than in several other organs/tissues (Callerio Foundation, data on file). This, however, is not sufficient to explain the significantly higher amount of ruthenium in the primary mass of B16 melanoma, and we must stress that most likely this effect depends on the characteristics of melanoma cells. Nevertheless, the relatively high ruthenium concentration in the primary tumor does not account for significant effects of NAMI-A on primary tumor growth, which is slightly reduced by approximately 20% compared with the untreated controls. It must be stressed that B16F10 melanoma cells show limited sensitivity to NAMI-A, and after cell exposure to 1 mM NAMI-A (a concentration close to that found in the primary tumor mass), only a 40% reduction of cell viability [determined in vitro with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test; data not reported] was found. One important consequence is that it is evident how, with a drug such as NAMI-A that has repeatedly demonstrated to be selectively effective on metastatic cells rather than on any kind of tumor cells (Sava et al., 2003; Bacac et al., 2004), in vitro cytotoxicity does not predict in vivo tumor reduction.

In addition, the effects on gelatinases appear to be even more complex. In this study, NAMI-A was shown to inhibit the active form of gelatinases released in the conditioned medium of in vitro treated cells, and it also decreased plasma gelatinase activity after in vivo treatment. This latter effect is in agreement with the proposed prognostic and diagnostic utility for MMP-2 and MMP-9 levels in plasma serum (Garbisa et al., 1992; Zucker et al., 1999; Ranuncolo et al., 2002) and might be correlated with the significant effect on lung metastasis reduction in the treated mice. Further studies may be necessary to confirm this. In the future, gelatinase plasma levels may be useful for forecasting tumor response to therapy with this class of compounds.

	Footnotes

 
This work was supported by contributions from Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2004-2005), and from Fondazione CRTrieste to the Metalli Anticancro Dell'Era postgenomica (MADE) project and to the Laboratorio per Identificare Nuovi Farmaci Antimetastasi laboratory.

doi:10.1124/jpet.105.095141.

ABBREVIATIONS: NAMI-A, imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate; MCa, murine mammary carcinoma; MMP, matrix metalloprotease; PBS, phosphate-buffered saline; MEM, minimum essential medium with Hanks' salt; BAEC, bovine aortic endothelial cell; FITC, fluorescein isothiocyanate.

Address correspondence to: Prof. G. Sava, Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7, 34127 Trieste, Italy. E-mail: g.sava{at}callerio.org

	References

Top Abstract Materials and Methods Results Discussion References

 

Albelda SM, Mette SA, Elder DE, Stewart R, Damjanovich L, Herlyn M, and Buck CA (1990) Integrin distribution in malignant melanoma: association of the ₃ subunit with tumor progression. Cancer Res 50: 6757–6764.[Abstract/Free Full Text]

Alessio E, Mestroni G, Bergamo A, and Sava G (2004a) Ruthenium anticancer drugs. Metal Ions Biol Syst 42: 323–351.

Alessio E, Mestroni G, Bergamo A, and Sava G (2004b) Ruthenium antimetastatic agents. Curr Top Med Chem 4: 1525–1535.[CrossRef][Medline]

Bacac M, Vadori M, Sava G, and Pacor S (2004) Cocultures of metastatic and host immune cells: selective effects of NAMI-A for tumor cells. Cancer Immunol Immunother 53: 1101–1110.[CrossRef][Medline]

Ballestrem C, Bernhard Wehrle-Haller B, Boris Hinz B, and Imhof BA (2000) Actin-dependent lamellipodia formation and microtubule-dependent tail retraction control-directed cell migration. Mol Biol Cell 11: 2999–3012.[Abstract/Free Full Text]

Bergamo A, Gagliardi R, Scarcia V, Furlani A, Alessio E, Mestroni G, and Sava G (1999) In vitro cell cycle arrest, in vivo action on solid metastasizing tumours and host toxicity of the anti-metastatic drug NAMI-A and of cisplatin. J Pharmacol Exp Ther 289: 559–564.[Abstract/Free Full Text]

Birch M, Mitchell S, and Hart IR (1991) Isolation and characterization of human melanoma cell variants expressing high and low levels of CD44. Cancer Res 51: 6660–6667.[Abstract/Free Full Text]

Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, Stetler-Stevenson WG, Quigley JP, and Cheresh DA (1996) Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3. Cell 85: 683–963.[CrossRef][Medline]

Clarke MJ (1989) Ruthenium chemistry pertaining to the design of anticancer agents, in Progress in Clinical Biochemistry and Medicine: Non-platinum Metal Complexes in Cancer Chemotherapy (Clarke MJ ed) pp 25–39, Springer-Verlag, Berlin.

Cocchietto M, Zorzet S, Sorc A, and Sava G (2003) Primary tumor, lung and kidney retention and antimetastasis effect of NAMI-A following different routes of administration. Investig New Drugs 21: 55–62.[CrossRef][Medline]

Danen EH, Ten Berge PJ, Van Muijen GN, Van't Hof-Grootenboer AE, Brocker EB, and Ruiter DJ (1994) Emergence of alpha 5 beta 1 fibronectinand alpha v beta 3 vitronectin-receptor expression in melanocytic tumour progression. Histopathology 24: 249–256.[Medline]

Deryugina EI, Bourdon MA, Luo GX, Reisfeld RA, and Strongin A (1997) Matrix metalloproteinase-2 activation modulates glioma cell migration. J Cell Sci 110: 2473–2482.[Abstract]

Drukala J, Rajwa B, Pietrzkowski Z, and Korohoda W (2003) Comparison of daunomycin influence upon human keratinocytes and melanoma HTB 140 cells. Image cytometry study. Anticancer Res 23: 419–423.[Medline]

Frausin F, Scarcia V, Cocchietto M, Furlani A, Serli B, Alessio E, and Sava G (2005) Free exchange across cells and echistatin-sensitive membrane target for the metastasis inhibitor NAMI-A on KB tumor cells. J Pharmacol Exp Ther 313: 227–233.[Abstract/Free Full Text]

Garbisa S, Scagliotti G, Masiero L, Di Francesco C, Caenazzo C, Onisto M, Micela M, Stetler-Stevenson WG, and Liotta LA (1992) Correlation of serum metalloproteinase levels with lung cancer metastasis and response to therapy. Cancer Res 52: 4548–4549.[Abstract/Free Full Text]

Herzog M, Draegar A, Ehler E, and Small JV (1994) Immunofluorescence microscopy of the cytoskeleton, in Cell Biology: A Laboratory Handbook (Celis JE ed) 2nd ed, vol 2, pp 355–360, Academic Press, NY.

Honn KV and Tang DG (1992) Adhesion molecules and tumor cell interaction with endothelium and subendothelial matrix. Cancer Met Rev 11: 353–375.[CrossRef][Medline]

Keppler BK, Henn M, Juhl UM, Berger MR, Niebl R, and Wagner FE (1989) New ruthenium complexes for the treatment of cancer, in Progress in Clinical Biochemistry and Medicine: Non-platinum Metal Complexes in Cancer Chemotherapy (Clarke MJ ed) pp 41–69, Springer-Verlag, Berlin.

Johnson JP (1991) Cell adhesion molecules of the immunoglobulin supergene family and their role in malignant transformation and progression to metastatic disease. Cancer Met Rev 10: 11–22.[CrossRef][Medline]

Li X, Regezi J, Ross FP, Blystone S, Ilic D, Leong SP, and Ramos DM (2001) Integrin _v3 mediates K1735 murine melanoma cell motility in vivo and in vitro. J Cell Sci 114: 2665–2672.

MacDougall JR, Bani MR, Lin Y, Rak J, and Kerbel RS (1995) The 92-kDa gelatinase B is expressed by advanced stage melanoma cells: suppression by somatic cell hybridization with early stage melanoma cells. Cancer Res 55: 4174–4181.[Abstract/Free Full Text]

Mestroni G, Alessio E, and Sava G (1998), inventors; Sigea S.r.l., assignee. New salt of anionic complexes of Ru(III) as antimetastatic and antineoplastic agents. International patent PCT C 07F 15/00, A61K 31/28. U.S. patent WO98/00431. 2001 Apr 24.

Rademaker-Lakhai JM, van den Bongard D, Pluim D, Beijnen JH, and Schellens JHM (2004) A phase I and pharmacological study with imidazolium-trans-DMSO-imidazole-tetrachlororuthenate, a novel ruthenium anticancer agent. Clin Cancer Res 10: 3717–3727.[Abstract/Free Full Text]

Ranuncolo SM, Matos E, Loria D, Vilensky M, Rojo R, Bal de Kier Joffè E, and Ines Puricelli L (2002) Circulating 92-kilodalton matrix metalloproteinase (MMP-9) activity is enhanced in the euglobulin plasma fraction of head and neck squamous cell carcinoma. Cancer 94: 1483–1491.[CrossRef][Medline]

Sava G, Capozzi I, Clerici K, Gagliardi G, Alessio E, and Mestroni G (1998) Pharmacological control of lung metastases of solid tumours by a novel ruthenium complex. Clin Exp Metastasis 16: 371–379.[CrossRef][Medline]

Sava G, Clerici K, Capozzi I, Cocchietto M, Gagliardi R, Alessio E, Mestroni G, and Perbellini A (1999a) Reduction of lung metastasis by ImH[trans-RuCl4(DMSO)Im]: mechanism of the selective action investigated on mouse tumors. Anticancer Drugs 10: 129–138.[CrossRef][Medline]

Sava G, Frausin F, Cocchietto M, Vita F, Podda E, Spessotto P, Furlani A, Scarcia V, and Zabucchi G (2004) Actin-dependent tumor cell adhesion after short exposure to the antimetastatic ruthenium complex NAMI-A. Eur J Cancer 40: 1383–1396.[CrossRef][Medline]

Sava G, Gagliardi R, Bergamo A, Alessio E, and Mestroni G (1999b) Treatment of metastases of solid mouse tumours by NAMI-A: comparison with cisplatin, cyclophosphamide and dacarbazine. Anticancer Res 19: 969–972.[Medline]

Sava G, Zorzet S, Turrin C, Vita F, Soranzo M, Zabucchi G, Cocchietto M, Bergamo A, DiGiovine S, Pezzoni G, et al. (2003) Dual action of NAMI-A in inhibition of solid tumor metastasis: selective targeting of metastatic cells and binding to collagen. Clin Cancer Res 9: 1898–1905.[Abstract/Free Full Text]

Seftor RE, Seftor EA, Gehlsen KR, Stetler-Stevenson WG, Brown PD, Ruoslahti E, and Hendrix MJ (1992) Role of the alpha v beta 3 integrin in human melanoma cell invasion. Proc Natl Acad Sci USA 89: 1557–1561.[Abstract/Free Full Text]

Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, and Boyd MR (1990) New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 82: 1107–1112.[Abstract/Free Full Text]

Stearns ME and Wang M (1992) Taxol blocks processes essential for prostate tumor cell (PC-3 ML) invasion and metastases. Cancer Res 52: 3776–3781.[Abstract/Free Full Text]

Sun W and Schuchter LM (2001) Metastatic melanoma. Curr Treat Options Oncol 2: 193–202.[Medline]

Tamura H and Arai T (1992) Determination of ruthenium in biological tissue by graphite furnace AAS after decomposition of the sample by tetramethylammonium hydroxide. Bunseki KagaKu 41: 13–17.

Tomasek JJ, Halliday NL, Updike DL, Ahern-Moore JS, Vu TK, Liu RW, and Howard EW (1997) Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton. J Biol Chem 272: 7482–7487.[Abstract/Free Full Text]

Trikha M, Timar J, Zacharek A, Nemeth JA, Cai Y, Dome B, Somlai B, Raso E, Ladanyi A, and Honn KV (2002) Role for ₃ integrin in human melanoma growth and survival. Int J Cancer 101: 156–167.[CrossRef][Medline]

Voura EB, Ramjeesingh RA, Montgomery AMP, and Siu CH (2001) Involvement of integrin _v3 and cell adhesion molecule L1 in transendothelial migration of melanoma cells. Mol Biol Cell 12: 2699–2710.[Abstract/Free Full Text]

Westerlund A, Hujanen E, Hoyhtya M, Puistola U, and Turpeenniemi-Hujanen T (1997) Ovarian cancer cell invasion is inhibited by paclitaxel. Clin Exp Metastasis 15: 318–328.[CrossRef][Medline]

Zorzet S, Bergamo A, Cocchietto M, Sorc A, Gava B, Alessio E, Iengo E, and Sava G (2000) Lack of in vitro cytotoxicity, associated to increased G(2)-M cell fraction and inhibition of matrigel invasion, may predict in vivo-selective antimetastasis activity of ruthenium complexes. J Pharmacol Exp Ther 295: 927–933.[Abstract/Free Full Text]

Zucker S, Hymowitz M, Conner C, Zarrabi HM, Hurewitz AN, Matrisian L, Boyd D, Nicolson G, and Montana S (1999) Measurement of matrix metalloproteinases and tissue inhibitors of metalloproteinases in blood and tissues. Clinical and experimental applications. Ann NY Acad Sci 878: 212–227.[Abstract/Free Full Text]

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