Inhibition of B 16 Melanoma Metastases with the Ruthenium Complex Imidazolium trans-Imidazoledimethylsulfoxide-tetrachlororuthenate and Down-Regulation of Tumor Cell Invasion

The antimetastatic ruthenium complex imidazolium transimidazoledimethylsulfoxide-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 integrin3 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 M 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. Melanoma is the most aggressive form of skin cancer. Despite recent advances, the results of chemotherapy for patients with metastatic melanoma remain unsatisfactory because of the relative drug resistance of metastatic cells (Drukala et al., 2003). The standard treatment for patients with metastatic melanoma has not been defined, and different chemotherapeutic agents have shown activity, although dacarbazine remains the reference agent. In addition, combination chemotherapy and biochemotherapy have been studied, but the response rate never exceeds 15 to 20% (Sun and Schuchter, 2001). 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). 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. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. 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. 0022-3565/06/3171-284–291$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 95141/3090976 JPET 317:284–291, 2006 Printed in U.S.A. 284 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from 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 integrin3 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 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 M NAMI-A in PBS Ca -Mg 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 l 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 l /ml streptomycin, 2 mM L-glutamine, 100 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 intrafootpad with 0.5 10/50 l 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 l/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 8m pore size were precoated with 5 l/50 l fibronectin on the reverse side and dried at room temperature. Matrigel (5 l/50 l; 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 M NAMI-A in PBS Ca -Mg 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 10/ml in MEM with 0.1% bovine serum albumin, 1% of 10 U/ml penicillin and 100 l/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