![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CHEMOTHERAPY, ANTIBIOTICS, AND GENE THERAPY
Department of Biomedical Sciences, University of Trieste, Trieste, Italy (S.P., S.Z., M.B., M.V., B.G., G.S.); and Callerio Foundation-Onlus, Institutes of Biological Research, Trieste, Italy (M.C., C.T., A.C., G.S.)
Received January 27, 2004; accepted April 6, 2004.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). This effect was always dissociated from a concomitant similar reduction of primary tumor growth and to any significant cytotoxic effect in vitro on human and on murine tumor cell lines up to millimolar concentrations (Bergamo et al., 2000; Pacor et al., 2001; Sava et al., 2003). These peculiarities make NAMI-A an interesting compound for treating human disseminated tumors with an as yet unknown mechanism of action but different from that of "classical" cytotoxic chemotherapeutic drugs. Recently, NAMI-A has completed a phase I clinical trial at the Netherlands Cancer Institute (Amsterdam, The Netherlands) on 24 patients, without any unexpected toxicity and a maximum tolerated dose at doses compatible with those active on metastases in preclinical models (J.H.M. Schellens, personal communication to G.S.).
Chemical studies pointed out the propensity of NAMI-A to give hydrolyzed species in physiological conditions, but only the unmodified molecule is capable to bind to DNA (Bacac et al., 2004). To expose tumor cells to the unmodified molecule of NAMI-A, and to highlight the role of DNA as preferential target for the selective antimetastatic activity, we performed an intratumoral (i.t.) treatment in a gel-carrier system with the aim of achieving in situ a concentration higher and for prolonged times than that obtained with systemic routes.
Recently, local administration (i.t.) of drugs has lead to a proliferation of research with the double aim of improving therapeutic outcomes and of limiting adverse systemic events (Goldberg et al., 2002). The great interest in i.t. drug administration is confirmed by the rising number of studies on animal models (Smith et al., 1999) and in clinical trials (Vogl et al., 2002). The therapeutic target of such site-specific treatment was a large variety of inoperable advanced malignant solid tumors. The drugs used for these kinds of treatment are usually incorporated into carriers such as purified bovine collagen gel matrix (Malhotra and Plosker, 2001), slow-release polymers (Jackson et al., 2000), or microspheres (Goldberg and Nakajima, 1980).
In addition to information on the efficacy of i.t. treatment against tumor growth and metastasis formation, the use of this route of administration also will provide more insight into the modality of action of this ruthenium compound that has always been given by systemic routes. More specifically, the aim of this study is to analyze the effects of NAMI-A on tumor cells and on tumor infiltrating lymphocytes (TILs) with particular attention to CD44 expression. CD44 family, a ubiquitous group of transmembrane adhesion glycoproteins, is involved in, e.g., cell-cell and cell-matrix interactions (Green et al., 1988; Lesley et al., 1993), lymphocyte homing and activation (DeGrendele et al., 1997; Gal et al., 2003), and transmission of growth signals. The external protein's domain acts mainly as a hyaluronic acid (HA) binder, whereas the cytoplasmic domain is bound to ankyrin, a cortex protein involved in the regulation of cell cytoskeleton (Bourguignon et al., 1995; Naot et al., 1997). Recent studies have demonstrated that CD44 is involved in the metastatic process at two crucial levels: cell adhesion to the extracellular matrix and cell motility (Herrera-Gayol and Jothy, 1999). Several studies have highlighted the possible use of CD44 expression level as a predictor/prognostic factor of metastatic potential (Xin et al., 2001; Yamaguchi et al., 2002; Wobus et al., 2002).
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The compound was dissolved in a solution of 1:1 volume ratio of isotonic apyrogenic saline and 50 µg/kg Matrigel, to reach a final Matrigel concentration of 25 µg/kg. NAMI-A was administered intratumorally at the dose of 35 mg/kg/day on days 10 to 15 from tumor implant, for 6 days of treatment. Control animals were treated in the same way with NAMI-A-free solution. We assumed that the subcutaneous tumor mass is physically identifiable with a solid derived from a rotation ellipsoid with a major and minor axis. Before treatment, the daily dose was divided into two aliquots of same volume that were administered on days 10, 12, and 14 at the two extremities of the major axis and on days 11, 13, and 15 at the two extremities of the minor axis after tumor implantation.
|
Tumor Model. The murine mammary carcinoma (MCa) was used. Briefly, tumor line was locally maintained by serial biweekly passages of 106 viable tumor cells, obtained from donors similarly inoculated 2 weeks before, and injected i.m. into the calf of the left hind leg of CBA/Lac female adult mice (Poliak-Blazi et al., 1981). Tumor propagation for experimental purpose was performed by subcutaneous implant.
Primary tumor measurement, metastasis count, and mass evaluation. Primary tumor growth was quantified with caliper measurements by determining two orthogonal axis and calculating tumor weight with the formula (
/6)·
2·
, with being the shorter and the longer axis, with d = 1. Tumor measures were taken every 2 days, on days 10 to 22 from tumor implant. Metastases were counted and measured, with graduated ocular, under a light microscope. The overall metastasis mass per mouse was calculated by applying the same formula used for the primary tumor to each single metastatic nodule. Number and mass of metastases were taken on day 22.
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).
Preparation of Samples for Atomic Absorption Spectroscopy. Lungs, liver, kidneys, primary tumor, blood (0.4-0.5 ml), and part of the tail were taken 24 and 168 h after the last administration (day 16 and 22, respectively, from tumor implant). A fragment of each tissue, and 50 µl of plasma, obtained from an aliquot of blood, were put in crio-vial, carefully weighed, and stored for ruthenium (Ru) quantitation analysis by means of atomic absorption spectroscopy. Samples were processed according to a modification of the procedures described by Tamura and Arai (1992). All specimens were dried in crio-vials. A first step of drying was performed overnight at 80°C, and a second step at 105°C, until the samples reached the constant dried weight. The decomposition of the dried samples was carried out with the addition of 100 µl of tetramethylammoniumhydroxide at 25% in water (Aldrich-Chimica, Milan, Italy) and 100 µl of MilliQ water directly into the vials. Volumes were adjusted to 0.5 ml with MilliQ water.
Ruthenium quantitation. The concentration of ruthenium in biological samples was measured in triplicate by means of graphite furnace atomic absorption spectrometry (model SpectrAA-300), supplied with a specific ruthenium emission lamp (hollow cathode lamp P/N 56-101447-00; Varian, Mulgrave, Victoria, Australia). Before each daily analysis session, a five-point calibration curve was traced using ruthenium custom-grade standard 998 mg ml-1 (Inorganic Ventures Inc., St. Louis, MO). To correct for possible deterioration of the graphite furnace during a daily working session, after every 12 samples, the calibration curve was retraced and a reslope standard was measured every six samples. The lower and higher limits of quantitation were set at the concentration levels that correspond, respectively, to the lowest and highest standard concentration used. The limit of detection was estimated according to the EURACHEM guide The Fitness for Purpose of Analytical Methods (http://www.eurachem.ul.pt). Lower limit of quantitation, higher limit of quantitation, and limit of detection were, respectively, 20, 100, and about 10 ng·Ru·ml-1 of sample. The quantitation of ruthenium was carried out in 10-µl samples at 349.9 nm with an atomizing temperature of 2500°C, using argon as purge gas at a flow rate of 3.0 l min-1. For further details concerning the furnace parameter settings, see Cocchietto and Sava (2000).
Measurement of Nitrite Formation. Nitric oxide production was evaluated quantifying nitrite (
) accumulation by a colorimetric assay according to the protocol of Steuhr and Nathan (1989). Aliquots of the samples were harvested from tumor suspension and allowed to react with nitrate reductase and NADPH+ and then with an equal volume of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylethylenediamine dihydrochloride in distilled water). The mixture was incubated in a flat-bottom 96-well culture plate for 10 min at room temperature. The absorbance was measured at 540 nm in a spectrophotometer. The values were obtained by comparison with standard concentrations of sodium nitrite.
MMP Activity Test. Cells (2 x 106) from the tumor homogenate were frozen, thawed, and then determination of protein content was done according to Bradford procedure. Cell samples were mixed with electrophoresis sample buffer [2% SDS, 10% glycerol, 50 mM Tris-HCl (pH 6.8), and 0.005% bromphenol blue], sonicated, and 80 µg of protein extract was 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 (6,500-205,00; Sigma-Aldrich, St. Louis, MO) was run on each gel. Densitometric analysis was done by Gel Compare II software.
Hemoglobin Quantitation. Hemoglobin was quantified in tumoral suspension and in blood using the Drabkin method (Drabkin and Austin, 1935) and Drabkin reagent kit 525 (Sigma-Aldrich).
Flow Cytometric Analyses. DNA staining of primary tumor mass. Cells (0.5 x 106) of primary tumor mass, obtained as described above, were stained with propidium iodide (PI) isotonic solution (0.5 mg of PI/ml H2O.) and left overnight before analysis.
CD44/PI staining of lymphocytes and tumor cells after density gradient separation. Tumor cells, obtained after gradient separation on Hystopaque, were stained with CD44-fluorescein isothiocyanate (FITC) and PI for a contemporaneous determination of cell membrane receptor and cell cycle. Briefly, 0.5 x 106 cells were stained with CD44-FITC (2 µg; Southern Biotechnology Associates, Birmingham, AL) for 60 min at 4°C in the dark and washed twice with phosphate-buffered saline-sodium azide-bovine serum albumin) (PBS-NaN3-BSA). The cells were then fixed with 1% paraformaldehyde for 15 min, washed twice with PBS, permeabilized with 100% methanol (added on vortex), and left for further 15 min. Methanol was removed by washing the cells twice with PBS and twice with PBS-NaN3-BSA-0.1% saponin. The last washing solution was maintained for 30 min. The cells were then resuspended in 0.5 ml of PBS containing 2.5 µg of PI, 4 µg/ml RNase and left overnight before analysis. All steps were performed at 4°C.
Circulating lymphocyte typing by monoclonal antibodies. Circulating lymphocytes were obtained from blood harvested by cardiac puncture from anesthetized animals. To avoid blood coagulation, 0.25 ml of EDTA (0.1 M) was used. Briefly, 25 µl of blood per animal was stained with fluorescent monoclonal antibodies (MoAbs) CD3-FITC, CD19-phycoerytreine (PE), CD4-FITC, CD8-PE, and CD44-FITC (all from Southern Biotechnology Associates) for 30 min at 4°C in the dark. Unbound antibodies were removed by washing twice with PBS-NaN3-BSA. Blood samples were then treated with NH4Cl solution for 5 min at room temperature to allow erythrocyte lysis. After washing with PBS-NaN3-BSA, the cells were resuspended with PBS. All steps were performed at 4°C and analyzed by flow cytometry. Aliquots treated with an irrelevant isotype matched and FITC/PE MoAb, run in parallel, were used to set the gates in the monoparametric histogram to include less than 2% aspecific fluorescence events. All flow cytometric analyses were performed with EPICS-XL2 (Beckman Coulter, Inc., Fullerton, CA). At least 10,000 events were acquired for each sample. Histograms were analyzed with the WinMDI software (J. Trotter, Scripps Research Institute, La Jolla, CA).
Scanning Electron Micrographs. Primary tumor mass, obtained as described above, was resuspended in PBS (20 x 106 cell/ml). A drop of each cell suspension was layered onto slides previously coated with poly-L-lysine solution (0.1 mg/ml) and allowed to adhere for 2 h at 4°C. Cells were then fixed with 2.5% glutaraldehyde at 4°C in 0.1 M cacodylate buffer overnight. Samples were then dehydrated in graded ethanol, vacuum dried, and mounted onto aluminum scanning electron micrograph (SEM) mounts. After sputter coating with gold, they were submitted for analysis with the Leica Stereoscan 430i instrument, at the Electronic Microscopy Section (Centro Servizi Polivalente d'Ateneo, University of Trieste, Trieste, Italy).
Statistical Analysis. Determinations of significant differences among groups were assessed by computerized program Instat (GraphPad Software Inc., San Diego, CA). Statistical tests used are reported in each table.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
The effects on metastases are shown on Table 1. Metastasis number is reduced to about one-third, and the whole metastatic mass undergoes a 6 to 7 times reduction versus controls, whereas the lungs of 60% of treated animals were free of macroscopically detectable metastases.
|
Ruthenium Quantitation. Ruthenium levels in tumor cell suspensions, lungs, kidney, liver, and plasma, after i.t. treatment, are listed in Table 2. Data represent concentrations of ruthenium measured, respectively, 24 and 168 h after last NAMI-A administration. Twenty-four hours after the end of treatment, the higher ruthenium concentration was found in tumor cell suspensions. After a further 6 days, this value decreased by about 1 order of magnitude. Compared with intratumor concentration, decrease of NAMI-A from lungs is markedly slower, suggesting a stronger binding of this ruthenium compound in this tissue.
|
Diploid/Aneuploid Cell Distribution inside Tumor Mass Suspension. PI staining of tumor cell suspensions, obtained from the tumor mass of untreated and of treated animals shows a different percentage of 2n and of aneuploid cells (Fig. 3A). Quantitative data are reported in Fig. 3B. The ratio of 2n versus aneuploid cells is about 1:1 in untreated animals and 3:1 in the group of animals treated with NAMI-A.
|
Effects on Hemoglobin Levels, Nitric Oxide Production, and MMP Activity. Data for hemoglobin quantification performed on peripheral blood and on tumor cell suspensions, at 24 and 168 h, and nitric oxide quantification performed at 168 h after the end of i.t. treatment, are reported in Table 3. No appreciable difference of hemoglobin concentration between treated and untreated animals is found in peripheral blood samples. Conversely, on tumor cell suspensions, a significant reduction (p < 0.05) of hemoglobin levels between treated and untreated animals is observed 168 h after the end of treatment. At this time, a significant accumulation of nitrites in the samples of tumors of the treated mice also is evidenced (p < 0.05).
|
Gelatin zymography assays on primary tumor extracts, harvested 168 h after the end of treatment, are reported in Fig. 4. Metalloprotease activity in samples of untreated controls (lane A) shows a degradation band at 92 kDa, corresponding to gelatinase MMP-9, whereas no appreciable gelatinolitic activity at 72 kDa is detectable, compared with the reference positive control cell line HT1080 (lane C). NAMI-A (lane B) reduces the gelatinolitic activity (MMP-9) of the tumor extracts by about 56%, based on results from densitometric analysis and as reported in Fig. 4, bottom.
|
CD44 Expression on Leukocytes. Table 4 shows the evaluation of CD44+ lymphocytes and of monocyte/polymorph-nucleated cells from peripheral blood of control and of NAMI-A-treated mice detected by flow cytometry analysis after staining with appropriate MoAb at 24 and 168 h from the end of treatment. NAMI-A increases the number of CD44+ lymphocytes by about 30% at any time of evaluation. No modifications of monocyte/polymorph-nucleated cells occur in the treated group compared with untreated controls.
|
CD44 Expression in Aneuploid Cells and in TILs. Flow cytometry analysis of CD44+ aneuploid and TIL cells, measured 24 and 168 h after the last NAMI-A injection, are reported in Fig. 5. At both 24 and 168 h, control samples (Fig. 5A) show a weak percentage of CD44+ diploid cells, and samples of NAMI-A-treated mice (Fig. 5B) do not significantly differ from controls. Conversely, control aneuploid cells show a relevant fraction of positive cells (above 35% of CD44+ over the total), a percentage that significantly drops in the NAMI-A-treated group (Fig. 5A). At 168 h, the percentage of CD44+ aneuploid cells of control animals rises to above 60%, whereas aneuploid cells in the treated animals remain low and comparable with those measured at 24 h.
|
SEM Analysis. SEM analysis of tumor cell suspensions from untreated animals shows MCa tumor cells characterized by a convolute membrane, with "lamellipodia" and "phylopodia", namely, "invadopodia" (Fig. 6A). This "aggressive" phenotype also correlates with the formation of homotypic aggregates (Fig. 6B). Conversely, ex vivo cells from NAMI-A-treated animals display a simpler membrane (Fig. 6C) and a significant interaction between leukocytes and tumor cells (Fig. 6D).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). This treatment is a suitable manner of maintaining longer exposure of tumor cells to nonhydrolyzed NAMI-A (Bacac et al., 2004).
Ruthenium concentration in host tissues, after i.t. treatment, is comparable with that obtained after systemic therapy, whereas in the primary tumor matrix, 24 h after last administration, it reaches a concentration of almost 1 order of magnitude higher than that in the other tissues analyzed. Six days later, intratumor ruthenium concentration drops to about one-tenth, still maintaining a value above the maximum level observed after i.p. treatment (Sava et al., 2002). The persistence of a high amount of NAMI-A in the tumor is responsible for the progressive reduction of primary tumor mass, statistically significant from 24 h after treatment onward. This effect is consistent with the reported in vitro antiproliferative threshold of NAMI-A on a number of tumor cell lines, which has been estimated to fall in the range of millimolar or greater concentrations (data on file). In the present work, this threshold is reached and maintained in the tumor mass for several days.
Nevertheless, this effect should not be simply attributed to cell cytotoxicity, because also in this condition, as reported in detail by Sava et al. (2003), NAMI-A is more active against cells endowed with metastatic ability based on results from a consistent number of animals free of macroscopically detectable colonies.
The unbalance between 2n/aneuploid ratio in the treated animals indicates that tumor reduction is due to tumor cell reduction paralleled by a consistent increase of TILs. The pronounced effect of NAMI-A on lung metastases, greater than that reported in previous work (Cocchietto et al., 2003), may be ascribed to the greater effect on primary tumor growth with a more complete elimination of the clone of the tumor cells endowed with metastatic ability (Sava et al., 2003).
NAMI-A effect on tumor cells seem to be attributable to its ability to modulate malignancy parameters such as CD44 expression. It is known that the increase of CD44+ tumor cells is associated to an enhanced HA binding, which in turn is related to cell migration and invasiveness (Bourguignon et al., 1981; Bartolazzi et al., 1994; Zhang et al., 1995). In our experimental conditions, NAMI-A significantly reduces the percentage of CD44+ tumor cells, and this reduction is present up to 7 days after the end of treatment.
Also invadopodia, i.e., cellular structures involved in tumor cell motility (Mueller and Chen, 1991; Monsky et al., 1994), are markedly reduced in tumor cells of the treated animals. Interestingly, these two markers show a close correlation, because CD44 is crucial to link the cortex protein ankyrin to the membrane-associated actomyosin contractile system, required for the formation of invadopodia and for the consequent tumor cell migration (Bourguignon et al., 1981). Reduction of invadopodia in tumor cells may also explain the decreased gelatinolitic activity of primary tumor, because it has been reported that MMP-9, in its proteolytically active form, is preferentially localized inside invadopodia (Mueller and Chen, 1991; Monsky et al., 1994). Thus, NAMI-A down-regulates CD44, reduces the formation of invadopodia, and down-modulates MMP-9 release, thereby interfering with the malignant properties of MCa tumor cells necessary to degrade the extracellular matrix and to allow tumor cell invasion and metastatic dissemination. These data are in agreement with the recent observations of the effects of NAMI-A on F-actin (Sava et al., 2004) and on 1-dependent contractile activity in aorta smooth muscle (M. Vadori, Ph.D. thesis data, Department of Biomedical Sciences, University of Trieste).
Another indication of modifications inside primary tumor, important for metastasis inhibition, is given by the significantly lower levels of hemoglobin induced by local treatment with NAMI-A, compared with untreated controls. These results are fully in agreement with the reported data of the effects of NAMI-A on EA.Hy926 (Vacca et al., 2002), ECV304 (Pintus et al., 2002; Sanna et al., 2002) endothelial cell lines, and in in vivo angiogenic systems (Vacca et al., 2002; Morbidelli et al., 2003). The overall data dealing with pronounced antiangiogenic effects correlated to the reduction of MMP release by cells inside primary tumor.
The appearance of numerous leukocytes into primary tumor mass and of CD44+ lymphocyte increase in the peripheral blood, seem to suggest a role for NAMI-A in promoting extravasation of blood lymphocytes throughout CD44 up-regulation. It is known that CD44-HA interaction, a pathway that mediates primary adhesion, can address lymphocytes to specific extra-lymphoid effector sites (DeGrendele et al., 1997). Once arrived into the primary tumor, lymphocytes down-regulate CD44 but increase nitric oxide production, as shown by the significant accumulation of nitrites in tumor cell suspensions obtained ex vivo from treated animals; an early burst of production of nitric oxide is reported as a sign of lymphocyte activation (Beltran et al., 2002).
These data strongly suggest that the antimetastatic effects of NAMI-A depend on the reduction of the malignant phenotype of tumor cells, and they are consistent with recent data indicating the stable and long-lasting effect of metastasis reduction for several transplant generations (Sava et al., 2003). This effect might be contributed by the potent action of NAMI-A on lymphocyte recruitment from circulation and to the consequent regulatory effects of these host cells on tumor growth and metastasis. NAMI-A i.t. administration seems thus an useful tool for neoadjuvant therapies, particularly where prognostic factors suggest high malignant tumors with elevated probability of metastatic dissemination.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: NAMI-A, imidazolium trans-imidazoledimethylsulfoxide tetrachlororuthenate, ImH[trans-RuCl4(DMSO)Im]; i.t., intratumor; TIL, tumor infiltrating lymphocyte; HA, hyaluronic acid; DMSO, dimethyl sulfoxide; MCa, murine mammary carcinoma; MoAb, monoclonal antibody; PI, propidium iodide; PBS-NaN3-BSA, phosphate-buffered saline-sodium azide-bovine serum albumin; PE, phycoerytreine; SEM, scanning electron micrograph.
Address correspondence to: Dr. Sabrina Pacor, Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7-9, 34127 Trieste, Italy. E-mail: pacorsab{at}univ.trieste.it
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Bacac M, Hotze A, van der Schilden K, Pacor S, Alessio E, Haasnoot J, Sava G, and Reedijk J (2004) NMR spectroscopy study of the coordination of the antimetastasis ruthenium complex [H2im]trans-Ru[RuCl4(DMSO)(Him)], NAMI-A to DNA model bases, correlation of its chemical stability with biological activity assessed by flow cytometry. J Inorg Biohem 98: 402-412.[CrossRef]
Bartolazzi A, Peach R, Aruffo A, and Stamenkovic I (1994) Interaction between CD44 and Hyaluronate is directly implicated in the regulation of tumor development. J Exp Med 180: 53-66.
Beltran B, Quintero M, Garcia-Zaragoza E, O'Connor E, Esplugues JV, and Moncada S (2002) Inhibition of mitochondrial respiration by endogenous nitric oxide: a critical step in Fas signaling. Proc Natl Acad Sci USA 99: 8892-8897.
Bergamo A, Zorzet S, Gava B, Sorc A, Alessio E, Iengo E, and Sava G (2000) Effects of NAMI-A and some related ruthenium complexes on cell viability after short exposure of tumor cells. Anticancer Drugs 11: 665-672.[CrossRef][Medline]
Bourguignon LY, Lida N, Welsh CF, Zhu D, Krongrad A, and Pasquale D (1995) Involvement of CD44 and its variant isoforms in membrane-cytoskeleton interaction, cell adhesion and tumor metastasis. J Neurooncol 26: 201-208.[CrossRef][Medline]
Bourguignon LYW, Zhu D, and Zhu H (1981) CD44 isoform-cytoskeleton interaction in oncogenic signaling and tumor progression. Front Biosci 3: 637-649.
Cocchietto M and Sava G (2000) Blood concentration and toxicity of the antimetastasis agent NAMI-A following repeated intravenous treatment in mice. Pharmacol Toxicol 87: 193-197.[CrossRef][Medline]
Cocchietto M, Zorzet S, Sorc A, and Sava G (2003) Relationship between in vivo distribution of NAMI-A to primary tumour, lungs and kidney following different routes of administration and metastasis inhibition. Investig New Drugs 21: 1-8.
DeGrendele HC, Estess P, and Siegelman MH (1997) Requirement for CD44 in activated T cell extravasation into an inflammatory site. Science (Wash DC) 278: 672-675.
Drabkin DL and Austin JH (1935) Spectrophotometric studies. II. Preparations from washed blood cells; nitric oxide hemoglobin and sulfhemoglobin. J Biol Chem 112: 51.
Gal I, Lesley J, Ko W, Stoop R, Hyman R, and Mikecz K (2003) Role of the extracellular and cytoplasmatic domains of CD44 in the rolling interaction of lymphoid cells with hyaluronan under physiologic flow. J Biol Chem 278: 11150-11158.
Goldberg EP, Hasdba AR, Almond BA, and Marotta JS (2002) Intratumoral cancer chemotherapy and immunotherapy: opportunities for nonsystemic preoperative drug delivery. J Pharm Pharmacol 54: 159-180.[CrossRef][Medline]
Goldberg EP and Nakajima A (1980) Biomedical Polymers: Polymeric Materials and Pharmaceuticals for Biomedical Use, 1st ed, Academic Press, New York.
Green SJ, Tarone G, and Underhill CB (1988) Aggregation of macrophages and fibroblasts is inhibited by a monoclonal antibody to the hyaluronate receptor. Exp Cell Res 178: 224-232.[CrossRef][Medline]
Herrera-Gayol A and Jothy S (1999) Adhesion proteins in the biology of breast cancer: contribution of CD44. Exp Mol Pathol 66: 149-156.[CrossRef][Medline]
Lesley J, Hyman R, and Kincade PW (1993) CD44 and its interaction with extracellular matrix. Adv Immunol 151: 271-335.
Jackson JK, Gleave ME, Yago V, Beraldi E, Hunter WL, and Burt HM (2000) The suppression of human prostate tumor growth in mice by the intratumoral injection of a slow-release polymeric paste formulation of paclitaxel. Cancer Res 60: 4146-4151.
Malhotra H and Plosker GL (2001) Cisplatin/epinephrine injectable gel. Drugs Aging 18: 787-793.[CrossRef][Medline]
Mestroni G, Alessio E, and Sava G (1998) New salts of anionic complexes of Ru(III), as antimetastatic and antineoplastic agents, PCT. C 07F 15/00, A61K 31/28. WO 98/00431 08.01.98.
Monsky WL, Lin CY, and Chen WT (1994) A potential marker protease of invasiveness, is localized on invadopodia of human malignant melanoma cells. Cancer Res 54: 5702-5710.
Morbidelli L, Donnini S, Filippi S, Messori L, Piccioli F, Orioli P, Sava G, and Ziche M (2003) Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers. Br J Cancer 88: 1484-1491.[CrossRef][Medline]
Mueller SC and Chen WT (1991) Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia. J Cell Sci 99: 213-225.
Naot D, Sionov RV, and Ish-Shalom D (1997) CD44: structure, function and association with the malignant process. Adv Cancer Res 71: 241-319.[Medline]
Pacor S, Vadori M, Vita F, Bacac M, Soranzo MR, Zabucchi G, and Sava G (2001) Isolation of a murine metastatic cell linee and preliminary test of sensitivity to the anti-metastasis agent NAMI-A. Anticancer Res 21: 2523-2530.[Medline]
Pintus G, Taddolini B, Posadino AM, Sanna B, Debidda M, Bernardini F, Sava G, and Ventura C (2002) Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down-regulates c-myc gene expression and endothelial cell proliferation. Eur J Biochem 269: 5861-5870.[Medline]
Poliak-Blazi M, Boranic M, Marzan B, and Radacic MA (1981) A transplantable aplastic mammary carcinoma of CBA mice. Vet Arh 51: 99-107.
Sanna B, Debidda M, Pintus G, Tadolini B, Posadino AM, Bennardini F, Sava G, and Ventura C (2002) The anti-metastatic agent imidazolium trans-imidazoledimethylsulfoxide-tetrachlororuthenate induces endothelial cell apoptosis by inhibiting the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway. Arch Biochem Biophys 403: 209-218.[CrossRef][Medline]
Sava G, Bergamo A, Zorzet S, Gava B, Casarsa C, Cocchietto M, Furlani A, Scarcia V, Serli B, Iengo E, et al. (2002) Influence of chemical stability on the activity of the antimetastasis ruthenium compound NAMI-A. Eur J Cancer 38: 427-435.
Sava G, Capozzi I, Clerici K, Gagliardi R, Alessio E, and Mestroni G (1998) Pharmacological control of lung metastases of solid tumors by a novel ruthenium complex. Clin Exp Metastasis 16: 371-379.[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-term exposure to the antimetastasis ruthenium complex NAMI-A Eur J Cancer 40: 1383-1396.
Sava G, Zorzet S, Turrin C, Vita F, Soranzio MR, Zabucchi G, Cocchietto M, Bergamo A, Di Giovine 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.
Smith JP, Kanekal S, Patawaran MB, Chen JY, Jones RE, Orenberg EK, and Yu NY (1999) Drug retention and distribution after intratumoral chemotherapy with fluorouracil/epinephrine injectable gel in human pancreatic cancer xenografts. Cancer Chemother Pharmacol 44: 267-274.[CrossRef][Medline]
Steuhr DJ and Nathan CT (1989) Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169: 1543-1555.
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.
Vacca A, Bruno M, Boccarelli A, Coluccia M, Ribatti D, Bergamo A, Garbisa S, Sartor L, and Sava G (2002) Inhibition of endothelial cell functions and of angiogenesis by the metastasis inhibitor NAMI-A. Br J Cancer 86: 993-998.[CrossRef][Medline]
Vogl TJ, Engelmann K, Mack MG, Straub R, Zangos S, Eichler K, Hochmuth K, and Orenberg E (2002) CT-guided intratumoral administration of cisplatin/epinephrine gel for treatment of malignant liver tumors. Br J Cancer 12: 524-529.
Wobus M, Rangwala R, Shein I, Hennigan R, Coila B, Lower EE, Yassin RS, and Sherman LS (2002) CD44 associates with EGFR and erbB2 in metastatizing mammary carcinoma cells. Appl Immunohistochem Mol Morphol 10: 34-39.[CrossRef][Medline]
Xin Y, Grace A, Gallagher MM, Curran BT, Leader MB, and Kay EW (2001) CD44V6 in gastric carcinoma: a marker of tumor progression. Appl Immunohistochem Mol Morphol 9: 138-142.[CrossRef][Medline]
Yamaguchi A, Goi T, Yu J, Hirono Y, Ishida M, Iida A, Kimura T, Takeuchi K, Katayama K, and Hirose K (2002) Expression of CD44v6 in advanced gastric cancer and its relationship to hematogenous metastasis and long-term prognosis. J Surg Oncol 79: 230-235.[CrossRef][Medline]
Zhang L, Underhill CB, and Chen LP (1995) Hyaluronan on the surface of tumor cells is correlated with metastatic behaviour. Cancer Res 55: 428-433.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
