Farnesyl Thiosalicylic Acid Chemosensitizes Human Melanoma In Vivo
2003; Elsevier BV; Volume: 120; Issue: 1 Linguagem: Inglês
10.1046/j.1523-1747.2003.12009.x
ISSN1523-1747
AutoresJulius Halaschek-Wiener, Yoel Kloog, Volker Wacheck, Burkhard Jansen,
Tópico(s)Cell Adhesion Molecules Research
ResumoMalignant melanoma is well known for its poor response to a variety of chemotherapeutic agents. Testing of numerous treatment strategies has identified dacarbazine as the most active single drug; however, its response rates in various clinical settings are quite limited. Defective apoptosis in combination with oncogenic proteins (such as activated Ras) in cell proliferation pathways plays a key part in both the development and disease progression of human melanoma. Farnesyl thiosalicylic acid, a novel Ras inhibitor, dislodges Ras proteins from the cell membrane, leading to inhibition of cell transformation and tumor growth. In this study we evaluated the effect of farnesyl thiosalicylic acid treatment on established human melanoma xenografts grown in mice with severe combined immunodeficiency as well as the chemosensitizing effect of farnesyl thiosalicylic acid in combination with dacarbazine. Daily administration of 10, 20, or 40 mg per kg of farnesyl thiosalicylic acid resulted in a concentration-dependent reduction in tumor growth, with growth inhibition reaching a mean value of 45±7%, at the highest concentration. The combination of farnesyl thiosalicylic acid (10 mg per kg per day) and dacarbazine (80 mg per kg per day) resulted in a significant reduction of 56%±9%, in mean tumor growth. Analysis of toxicologic parameters (mouse weight, blood cell counts, and blood chemistry) showed an acceptable and similar toxicity profile for both the single-agent farnesyl thiosalicylic acid treatment and the combination of farnesyl thiosalicylic acid plus dacarbazine treatment. Given the observed preclinical treatment responses and the low toxicity, our results support the notion that farnesyl thiosalicylic acid in combination with dacarbazine may qualify as a rational treatment approach for human melanoma. Malignant melanoma is well known for its poor response to a variety of chemotherapeutic agents. Testing of numerous treatment strategies has identified dacarbazine as the most active single drug; however, its response rates in various clinical settings are quite limited. Defective apoptosis in combination with oncogenic proteins (such as activated Ras) in cell proliferation pathways plays a key part in both the development and disease progression of human melanoma. Farnesyl thiosalicylic acid, a novel Ras inhibitor, dislodges Ras proteins from the cell membrane, leading to inhibition of cell transformation and tumor growth. In this study we evaluated the effect of farnesyl thiosalicylic acid treatment on established human melanoma xenografts grown in mice with severe combined immunodeficiency as well as the chemosensitizing effect of farnesyl thiosalicylic acid in combination with dacarbazine. Daily administration of 10, 20, or 40 mg per kg of farnesyl thiosalicylic acid resulted in a concentration-dependent reduction in tumor growth, with growth inhibition reaching a mean value of 45±7%, at the highest concentration. The combination of farnesyl thiosalicylic acid (10 mg per kg per day) and dacarbazine (80 mg per kg per day) resulted in a significant reduction of 56%±9%, in mean tumor growth. Analysis of toxicologic parameters (mouse weight, blood cell counts, and blood chemistry) showed an acceptable and similar toxicity profile for both the single-agent farnesyl thiosalicylic acid treatment and the combination of farnesyl thiosalicylic acid plus dacarbazine treatment. Given the observed preclinical treatment responses and the low toxicity, our results support the notion that farnesyl thiosalicylic acid in combination with dacarbazine may qualify as a rational treatment approach for human melanoma. S-transtrans farnesylthiosalicylic acid farnesyl transferase inhibitors severe combined immunodeficiency Malignant melanoma is one of the most rapidly increasing malignancies in the white population and has a mortality rate surpassed only by lung cancer (Slominski et al., 2001Slominski A. Wortsman J. Carlson A.J. Matsuoka L.Y. Balch C.M. Mihm M.C. Malignant melanoma: an update.Arch Pathol Lab Med. 2001; 125: 1295-1306PubMed Google Scholar). Age-adjusted incidence rates are 12 per 100,000 in the U.S.A. and about three times higher in some geographic areas, such as Australia (Helmbach et al., 2001Helmbach H. Rossmann E. Kern M.A. Schadendorf D. Drug-resistance in human melanoma.Int J Cancer. 2001; 93: 617-622Crossref PubMed Scopus (154) Google Scholar). Malignant melanoma is a tumor derived from activated or genetically altered melanocytes as a result of complex interactions between genetic, constitutional, and environmental factors (Slominski et al., 1998Slominski A. Wortsman J. Nickoloff B. McClatchey K. Mihm M.C. Ross J.S. Molecular pathology of malignant melanoma.Am J Clin Pathol. 1998; 110: 788-794PubMed Google Scholar, Slominski et al., 2001Slominski A. Wortsman J. Carlson A.J. Matsuoka L.Y. Balch C.M. Mihm M.C. Malignant melanoma: an update.Arch Pathol Lab Med. 2001; 125: 1295-1306PubMed Google Scholar;Halpern and Altman, 1999Halpern A.C. Altman J.F. Genetic predisposition to skin cancer.Curr Opin Oncol. 1999; 11: 132-138Crossref PubMed Scopus (33) Google Scholar). Early, localized melanoma has a good prognosis after adequate surgical therapy. The prognosis of metastatic melanoma, however, is poor despite the availability of a growing number of biologic, chemotherapeutic, and combined modality treatments. Today, dacarbazine is the most widely used single-agent chemotherapy for metastatic melanoma, but few patients obtain durable responses, and the long-term survival benefit from this drug is limited (Middleton et al., 2000Middleton M.R. Grob J.J. Aaronson N. et al.Randomized phase III study of temozolomide versus dacarbazine in the treatment of patients with advanced metastatic malignant melanoma.J Clin Oncol. 2000; 18: 158-166Crossref PubMed Google Scholar) due to the exceptionally high degree of drug resistance of this malignancy (Helmbach et al., 2001Helmbach H. Rossmann E. Kern M.A. Schadendorf D. Drug-resistance in human melanoma.Int J Cancer. 2001; 93: 617-622Crossref PubMed Scopus (154) Google Scholar). Dacarbazine, a methylating agent, requires metabolic activation in the liver through an N-demethylation reaction by the CYP450 system, and subsequently undergoes spontaneous cleavage to yield a stable metabolite (AIAC) and an active methylating species, diazomethane (Chabner et al., 1996Chabner B.A. Allegra C.J. Curt G.A. Cameron J.L. Antineoplastic agents.in: Hardman J.G. Limbird L.E. Goodman & and Gilman's. The Pharmacological Basis of Therapeutics. 9th edn. McGraw-Hill Inc., New York1996: 1233-1287Google Scholar;Reid et al., 1999Reid J.M. Kuffel M.J. Miller J.K. Rios R. Ames M.M. Metabolic activation of dacarbazine by human cytochromes P450: the role of CYP1A1, CYP1A2, and CYP2E1.Clin Cancer Res. 1999; 5: 2192-2197PubMed Google Scholar). Therefore, dacarbazine has no in vitro activity and is suitable only in in vivo models. In an attempt to improve chemotherapy response rates in malignant melanoma, numerous clinical trials have been performed using chemotherapy (with or without inclusion of dacarbazine) as well as immunotherapy (with interferons±interleukin-2), gene therapy, or combinatorial strategies. Unfortunately, it remains unclear whether any of these combination therapies is superior to single-agent dacarbazine in this disease (reviewed inHuncharek et al., 2001Huncharek M. Caubet J.F. McGarry R. Single-agent dacarbazine versus combination chemotherapy with or without immunotherapy in metastatic melanoma: a meta-analysis of 3273 patients from 20 randomized trials.Melanoma Res. 2001; 11: 75-81Crossref PubMed Scopus (140) Google Scholar). Thus, new treatment approaches are still being sought. Although a multitude of factors have been suspected of participating in melanoma growth, progression, and chemoresistance, the most common specific gene defects identified in melanoma are activating mutations in Ras genes (Van't Veer et al., 1989Van't Veer L.J. Burgering B.M. Versteeg R. et al.N-ras mutations in human cutaneous melanoma from sun-exposed body sites.Mol Cell Biol. 1989; 9: 3114-3116Crossref PubMed Scopus (246) Google Scholar;Ball et al., 1994Ball N.J. Yohn J.J. Morelli J.G. Norris D.A. Golitz L.E. Hoeffler J.P. Ras mutations in human melanoma: a marker of malignant progression.J Invest Dermatol. 1994; 102: 285-290Abstract Full Text PDF PubMed Google Scholar). The incidence of Ras gene mutations in human melanoma is about 20% (Ball et al., 1994Ball N.J. Yohn J.J. Morelli J.G. Norris D.A. Golitz L.E. Hoeffler J.P. Ras mutations in human melanoma: a marker of malignant progression.J Invest Dermatol. 1994; 102: 285-290Abstract Full Text PDF PubMed Google Scholar;van-Elsas et al., 1996van-Elsas A. Zerp S.F. van-der-Flier S. et al.Relevance of ultraviolet-induced N-ras oncogene point mutations in development of primary human cutaneous melanoma.Am J Pathol. 1996; 149: 883-893PubMed Google Scholar), representing mainly alterations in N-Ras at codon 61, whereas alterations in H-Ras and Ki-Ras are relatively rare (Van't Veer et al., 1989Van't Veer L.J. Burgering B.M. Versteeg R. et al.N-ras mutations in human cutaneous melanoma from sun-exposed body sites.Mol Cell Biol. 1989; 9: 3114-3116Crossref PubMed Scopus (246) Google Scholar;Ball et al., 1994Ball N.J. Yohn J.J. Morelli J.G. Norris D.A. Golitz L.E. Hoeffler J.P. Ras mutations in human melanoma: a marker of malignant progression.J Invest Dermatol. 1994; 102: 285-290Abstract Full Text PDF PubMed Google Scholar;van-Elsas et al., 1995van-Elsas A. Zerp S. van-der-Flier S. Kruse-Wolters M. Vacca A. Ruiter D.J. Schrier P. Analysis of N-ras mutations in human cutaneous melanoma: tumor heterogeneity detected by polymerase chain reaction/single-stranded conformation polymorphism analysis.Recent Results Cancer Res. 1995; 139: 57-67Crossref PubMed Scopus (39) Google Scholar). Because Ras proteins are regulators of multiple signaling pathways that control cell growth, differentiation, and apoptosis (Boguski and McCormick, 1993Boguski M.S. McCormick F. Proteins regulating Ras and its relatives.Nature. 1993; 366: 643-654Crossref PubMed Scopus (1725) Google Scholar;Marshall, 1996Marshall C.J. Ras effectors.Curr Opin Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (462) Google Scholar;Bos, 1997Bos J.L. Ras-like GTpases.Biochim Biophys Acta Rev Cancer. 1997; 1333: M19-M31Crossref PubMed Scopus (132) Google Scholar), the deregulation of other cellular factors may also mimic effects of aberrant Ras function even in the absence of a Ras mutation. Thus, the influence of normal and aberrant Ras function on the biology of human melanoma may be much greater than expected from the frequency of Ras gene mutations in this tumor. This notion further highlights the potential benefits that Ras inhibitors may provide in attempts to block the growth of malignant melanoma. Improved understanding of the molecular mechanisms of Ras processing and membrane targeting provided an important background for the development of compounds that would inhibit the association of Ras with the inner cell membrane. Such compounds include farnesyltransferase inhibitors (FTI), as well as inhibitors of the prenyl-CAAX protease, inhibitors of the methyltransferase, possible inhibitors of Ras trafficking, and inhibitors such as S-trans, trans farnesyl thiosalicylic acid (FTS) that interfere with the association of mature Ras with the cell membrane. FTI, the most widely studied group of Ras inhibitors, fail to inhibit the functions of N-Ras and K-Ras proteins, as both of these isoforms can be geranyl-geranylated under conditions where the farnesyl transferase is inhibited. These alternatively prenylated Ras isoforms are still targeted to the cell membrane and remain active (Kloog et al., 1999Kloog Y. Cox A.D. Sinensky M. Concepts in Ras-directed therapy.Expert Opin Invest Drugs. 1999; 8: 2121-2140Crossref PubMed Scopus (53) Google Scholar). The farnesyl group, which is common to all Ras proteins, may act as part of a recognition unit for specific anchorage lipids or protein(s) that interact with Ras in the cell membrane (Cox and Der, 1997Cox A.D. Der C.J. Farnesyltransferase inhibitors and cancer treatment: targeting simply Ras?.Biochim Biophys Acta. 1997; 1333: F51-F71PubMed Google Scholar). On the assumption that Ras functions might be inhibited by competitively displacing the mature protein from its putative membrane-anchorage domains, a series of organic compounds resembling the farnesylcysteine of Ras proteins was designed (Marciano et al., 1995Marciano D. Ben-Baruch G. Marom M. Egozi Y. Haklai R. Kloog Y. Farnesyl derivatives of rigid carboxylic acids-inhibitors of ras-dependent cell growth.J Med Chem. 1995; 38: 1267-1272Crossref PubMed Scopus (110) Google Scholar;Aharonson et al., 1998Aharonson Z. Gana-Weisz M. Varsano T. Haklai R. Marciano D. Kloog Y. Stringent structural requirements for anti-Ras activity of S-prenyl analogues.Biochim Biophys Acta. 1998; 1406: 40-50Crossref PubMed Scopus (52) Google Scholar). One of these compounds, FTS, was found to be a potent growth inhibitor of H-Ras-transformed Rat-1 (EJ) fibroblasts, specifically affecting the membrane-bound H-Ras protein in these cells (Marom et al., 1995Marom M. Haklai R. Ben-Baruch G. Marciano D. Egozi Y. Kloog Y. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid.J Biol Chem. 1995; 270: 22263-22270Crossref PubMed Scopus (169) Google Scholar;Haklai et al., 1998Haklai R. Weisz M.G. Elad G. et al.Dislodgment and accelerated degradation of Ras.Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (180) Google Scholar). Specificity of FTS towards Ras was suggested by the finding that FTS can inhibit the growth of fibroblasts transformed by ErbB2 acting upstream of Ras, but not the growth of cells transformed by v-Raf (which, unlike Raf-1, acts independently of Ras). FTS does not inhibit the farnesylation of H-Ras; it affects H-Ras–membrane interactions both in vitro and in vivo, dislodging the protein from its anchorage domains, thus facilitating its degradation and hence reducing the total amount of cellular Ras (Marom et al., 1995Marom M. Haklai R. Ben-Baruch G. Marciano D. Egozi Y. Kloog Y. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid.J Biol Chem. 1995; 270: 22263-22270Crossref PubMed Scopus (169) Google Scholar;Haklai et al., 1998Haklai R. Weisz M.G. Elad G. et al.Dislodgment and accelerated degradation of Ras.Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (180) Google Scholar). On the basis of these proposed general mechanisms, the therapeutic potential of FTS was tested in vitro in several cell lines of different origin. FTS was found to dislodge Ras proteins in all cells investigated so far, including H-Ras-, K-Ras-, and N-Ras-transformed rat fibroblasts (Marom et al., 1995Marom M. Haklai R. Ben-Baruch G. Marciano D. Egozi Y. Kloog Y. Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid.J Biol Chem. 1995; 270: 22263-22270Crossref PubMed Scopus (169) Google Scholar;Haklai et al., 1998Haklai R. Weisz M.G. Elad G. et al.Dislodgment and accelerated degradation of Ras.Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (180) Google Scholar;Jansen et al., 1999Jansen B. Schlagbauer-Wadl H. Kahr H. et al.Novel Ras antagonist blocks human melanoma growth.Proc Natl Acad Sci USA. 1999; 96: 14019-14024Crossref PubMed Scopus (88) Google Scholar), Rat-1 cells (Haklai et al., 1998Haklai R. Weisz M.G. Elad G. et al.Dislodgment and accelerated degradation of Ras.Biochemistry. 1998; 37: 1306-1314Crossref PubMed Scopus (180) Google Scholar), PC-12 cells (Haring et al., 1998Haring R. Fisher A. Marciano D. et al.Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion.J Neurochem. 1998; 71: 2094-2103Crossref PubMed Scopus (120) Google Scholar), Panc-1 cells (Weisz et al., 1999Weisz B. Giehl K. Gana-Weisz M. et al.A new functional Ras antagonist inhibits human pancreatic tumor growth in nude mice.Oncogene. 1999; 18: 2579-2588Crossref PubMed Scopus (103) Google Scholar), and colon carcinoma cells (Halaschek-Wiener et al., 2000Halaschek-Wiener J. Wacheck V. Schlagbauer-Wadl H. Wolff K. Kloog Y. Jansen B. A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells.Mol Med. 2000; 6: 693-704PubMed Google Scholar). Furthermore, recent experiments showed that FTS reduces the amounts of activated N-Ras and wild-type Ras isoforms in both human melanoma cells and Rat-1 fibroblasts, blocks the activity of the Ras-dependent extracellular signal-regulated kinase in melanoma cells, and inhibits the growth of N-Ras-transformed fibroblasts and human melanoma cells in vitro and reverses their transformed phenotype. FTS also inhibits the growth of human melanoma cells injected into mice with severe combined immunodeficiency (SCID) without evidence of drug-related toxicity (Jansen et al., 1999Jansen B. Schlagbauer-Wadl H. Kahr H. et al.Novel Ras antagonist blocks human melanoma growth.Proc Natl Acad Sci USA. 1999; 96: 14019-14024Crossref PubMed Scopus (88) Google Scholar). In addition to its Ras-dislodging property, FTS upregulates the tumor suppressor protein p53 in colon cancer cells, leading to cell cycle arrest in G0/G1 via the p53/p21(waf1/cip1) signaling pathway (Halaschek-Wiener et al., 2000Halaschek-Wiener J. Wacheck V. Schlagbauer-Wadl H. Wolff K. Kloog Y. Jansen B. A novel Ras antagonist regulates both oncogenic Ras and the tumor suppressor p53 in colon cancer cells.Mol Med. 2000; 6: 693-704PubMed Google Scholar). Taken together, these findings support the notion that a change in the Ras-signaling cascade leads to complex restructuring of the general survival strategies of human cancer cells; however, H-Ras-transformed Rat-1 fibroblasts, Panc-1, and SW480 cells exposed to high concentrations of the Ras inhibitor FTS for prolonged periods (Gana-Weisz et al., 2002Gana-Weisz M. Halaschek-Wiener J. Jansen B. Elad G. Haklai R. Kloog Y. The Ras Inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance.Clin Cancer Res. 2002; 8: 555-565PubMed Google Scholar) did not establish resistance against the drug. Furthermore, it was shown that both Panc-1 and SW480 cancer cells were highly chemosensitized to gemcitabine and doxorubicin in vitro. Chemosensitization of Panc-1 tumors by FTS was also demonstrated in nude mice, with a synergistic effect on the survival rate of the combined treatment of gemcitabine and FTS (Gana-Weisz et al., 2002Gana-Weisz M. Halaschek-Wiener J. Jansen B. Elad G. Haklai R. Kloog Y. The Ras Inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance.Clin Cancer Res. 2002; 8: 555-565PubMed Google Scholar). On the basis of these observations, it seemed reasonable to speculate that FTS might chemosensitize human melanoma in vivo. In this study we used FTS in a dacarbazine-based combination chemotherapy in a human melanoma SCID mouse xenotransplantation model. We evaluated the effects of both single-agent treatment with FTS or dacarbazine and combinations of FTS and dacarbazine on established melanoma xenografts. FTS inhibited tumor growth in a concentration-dependent manner and chemosensitized melanomas harboring an activated N-Ras gene. Analysis of toxicologic parameters yielded a highly favorable toxicity profile for both single-agent FTS and combined treatment with FTS and dacarbazine. Clinical grade FTS and CRYSMEB (cyclodextrin) were obtained from Thyreos Corporation (Newark, NJ). Dacarbazine was from MEDAC (Hamburg, Germany). FTS was formulated in CRYSMEB, according to the manufacturer's instructions, to increase its solubility by covering the hydrophobic farnesyl moiety. The stock solution of phosphate-buffered saline (PBS; Gibco, Carlsbad, CA) containing FTS in CRYSMEB (10% wt/wt) was stable for the duration of the experiments. CRYSMEB in PBS served as a control ("carrier control") in all experiments. Dacarbazine was dissolved in sterile H2O to a final concentration of 10 mg per ml and the required amount of dacarbazine was injected intraperitoneally. The human melanoma cell line 607B was obtained from Dr Peter Schrier, Leiden, the Netherlands. These cells harbor a naturally occurring N-Ras gene mutation (van-Elsas et al., 1995van-Elsas A. Zerp S. van-der-Flier S. Kruse-Wolters M. Vacca A. Ruiter D.J. Schrier P. Analysis of N-ras mutations in human cutaneous melanoma: tumor heterogeneity detected by polymerase chain reaction/single-stranded conformation polymorphism analysis.Recent Results Cancer Res. 1995; 139: 57-67Crossref PubMed Scopus (39) Google Scholar). Cells were cultured in Dulbecco minimal Eagle's medium (Gibco) supplemented with 10% fetal calf serum (Gibco) in a humidified 5% CO2/95% ambient air atmosphere at 37°C. The cells needed for the experiments were grown in culture flasks, harvested on the day of injection, counted, and diluted in sterile PBS to a final concentration of 1×107 cells per 200 μl. Pathogen-free female C.B-17 scid/scid (SCID) mice, 4–6 wk old, tested for leakiness, were obtained from Harlan-Winkelmann (Borchen, Germany). Mice were housed in laminar flow racks and microisolator cages under specific pathogen-free conditions and received autoclaved food and water. SCID mice were injected subcutaneously (s.c.) into the left lower flank with 1×107 (200 μl) human melanoma cells (607B). One week after cell inoculation, when palpable tumors were established (0.5–0.7 mm3), mice were randomized into the respective groups (n=8 in each group) and treatment was initiated. FTS (treatment), carrier solution (CRYSMEB), or physiologic NaCl solution (saline control) was administered daily for 4 wk via the intraperitoneal (i.p.) route at 10, 20, or 40 mg FTS per kg. In a combination therapy experiment FTS (10 mg per kg) was injected i.p. daily for 2 wk, and dacarbazine (80 mg per kg) was injected i.p. on 5 consecutive days starting on day 1 of FTS treatment. This treatment regimen was followed by a 2 wk observation period. Tumor size was measured twice a week during treatment and during the observation period. Tumor volumes were calculated as follows: V (mm3)=(length×width2)×0.5. Four weeks after the start of treatment the mice were killed, the tumors were isolated, and body and tumor weights were recorded. The results (tumor weights) are presented as the mean tumor weights (mean±SD) of eight individual mice. Whole blood for blood cell analysis was withdrawn from mice in the combination chemotherapy study. All animal studies were approved by the University of Vienna Animal Welfare Committee. Statistical significance of differences in mean tumor weights (in FTS-treated or combination-treated mice compared with controls) was determined using the Mann–Whitney U-test. Multiple comparisons were calculated using one-way ANOVA, and post-tests among treatment groups were carried out using the Scheffé test (SPSS, release 10.0.7, Chicago, IL). p<0.05 was considered to be statistically significant. Tumor-free mice of the same type as the experimental mice were used to investigate the toxicity profile of FTS (20 mg per kg) with and without dacarbazine treatment (80 mg per kg). Mice treated with single-agent FTS or single-agent dacarbazine (n=8 per group) were injected with the respective drugs for 3 d and were killed on day 4. Mice receiving the drugs in combination were treated for 4 d. Mice that received FTS plus dacarbazine combination treatment (n=8 per day) were killed on days 3, 4, and 5 after the initial drug application. Their bodies were weighed and whole blood for blood cell analysis (n=4) and serum for enzyme activity assays (n=4) was obtained. For analysis of blood cells, 0.5–0.8 ml of whole blood was withdrawn (n=4 from each group) in syringes rinsed with heparin (Baxter, Deerfield, IL). Concentrations of white and red blood cells and platelets were determined by fluorescence-activated cell sorter analysis (FACStar, Becton-Dickinson, San Jose, CA). Serum was isolated by centrifugation at 800 g. in an Eppendorf centrifuge at 4°C after incubation for 2 h at 4°C, and analyzed for liver, pancreas, and kidney function by standard laboratory procedures. The main purpose of this study was to evaluate the therapeutic potential and the toxicity of single-agent FTS treatment as well as its chemosensitizing effect when administered in combination with dacarbazine. First we evaluated the therapeutic effect of FTS on established xenografts grown subcutaneously. In all the xenografted mice, xenotransplanted 607B cells grew as localized tumors and no evidence of metastasis was observed. We were unable to detect any metastasizing 607B melanoma cells in the main organs removed from the mice at the end of the experiments (data not shown). Single-agent FTS treatment was initiated 1 wk after cell inoculation, when small tumors (0.5–0.7 mm3) were palpable. Experimental animals were treated with 10, 20, or 40 mg FTS per kg or with the carrier (CRYSMEB) or saline (physiologic NaCl) control solutions (Figure 1). After 4 wk of daily treatment, the mice were killed and the mean tumor weights (n=8 in each group) were recorded. As shown in Figure 1(a), we observed a concentration-dependent decrease in tumor size. Carrier control treatment had only a small effect on tumor growth (the mean value was –11%±2%); the FTS-treated groups showed concentration-dependent growth reduction of 31%±5%, 38%±8%, and 45%±7% at 10, 20, and 40 mg FTS per kg, respectively, relative to saline-treated controls. FTS treatment at 40 mg per kg resulted in significant growth inhibition compared with both the carrier (p<0.01) and saline (p<0.001) control groups, whereas mice in the groups treated with 10 or 20 mg FTS per kg showed significant reductions in xenograft growth compared with the saline control (p<0.01) but not to the carrier control group. Actual mean tumor weights (n=8 mice in each group) were 1.9 g±0.17 g, for the saline controls, 1.7 g±0.56 g for the carrier controls, and 1.3 g±0.22 g, 1.2 g±0.19 g, and 1.0 g±0.16 g for the groups treated with FTS at 10, 20, and 40 mg per kg, respectively (Figure 1a). These results clearly demonstrate concentration-dependent inhibition of mean tumor growth by FTS. Monitoring of body weight as an indicator of toxic side-effects revealed a nonsignificant reduction in mean body weight at the highest dose used (-14%±2% in the 40 mg FTS per kg group) (Figure 2a). FTS reduced tumor growth in a time- and concentration-dependent manner (Figure 1b): mice treated with FTS at 40 mg per kg clearly displayed the smallest xenografts, a finding that was noticeable as early as day 11. Only the xenografts of mice treated with FTS at 20 mg per kg were of similar size until day 21 (Figure 1b).Figure 2Body weight assessment as a toxicologic indicator. Mouse body weight (mean±SD, n=8) of mice was recorded at the end of single-agent FTS treatment (A) or combination treatment (B) experiments to determine the toxic effects of these treatment regimens. Neither the FTS treatment shown in A nor the treatments (dacarbazine alone or in combination with FTS) shown in B yielded a significant toxicologic effect as indicated by altered body weights.View Large Image Figure ViewerDownload (PPT) In view of these findings, we carried out follow-up experiments combining dacarbazine chemotherapy with FTS to evaluate the chemosensitization potential of the Ras antagonist. In the first experiment, single-agent treatment with FTS at 10 mg per kg per day led to a small reduction in tumor growth, which was statistically nonsignificant compared with the carrier control. A similar concentration (14 mg per kg per day) was previously shown to chemosensitize effectively human pancreatic carcinoma xenotransplants (Gana-Weisz et al., 2002Gana-Weisz M. Halaschek-Wiener J. Jansen B. Elad G. Haklai R. Kloog Y. The Ras Inhibitor S-trans,trans-farnesylthiosalicylic acid chemosensitizes human tumor cells without causing resistance.Clin Cancer Res. 2002; 8: 555-565PubMed Google Scholar). In initial toxicologic studies we determined the toxicity of single-dose dacarbazine administration by injecting one dose of 200 mg per kg or 400 mg dacarbazine per kg (i.p.) into SCID mice. Dacarbazine at 400 mg per kg was lethal, killing mice within 3 d of injection. Mice injected with 200 mg dacarbazine per kg showed clear but acceptable signs of toxicity. Combination treatment of 10 or 20 mg FTS per kg (daily for 14 d) and 200 mg dacarbazine per kg (once on day 8 of FTS treatment) yielded high overall toxicity and resulted in only marginal reduction of tumor growth (data not shown). As a dosage of 80 mg dacarbazine per kg administered on 5 consecutive days was previously shown by our group to be well tolerated and highly active in a SCID mouse model (Jansen et al., 1998Jansen B. Schlagbauer-Wadl H. Brown B.D. et al.bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice.Nat Med. 1998; 4: 232-234Crossref PubMed Scopus (456) Google Scholar), we adapted this regimen and investigated the anti-tumor effect of 10 mg FTS per kg given daily for 2 wk in combination with 80 mg dacarbazine per kg given on days 1–5 of FTS injection. Mice treated with carrier, carrier plus dacarbazine, single-agent dacarbazine, or saline were used as controls. The shortened FTS treatment period of only 2 wk elicited no response in the 10 mg FTS per kg group compared with the carrier control (Figure 3a). Tumors (n=8) in both groups showed an equal mean reduction of 20% (1.4 g±0.31 g, for 10 mg FTS per kg and 1.4 g±0.26 g, for the carrier) compared with xenografts in the saline-treated group. Tumors
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