Publicationof the InternationalUnion Against Cancer Publicationde I'Union InternationaleContre 1e Cancer Int. J. Cancer: 65,870-874 (1996) 0 1996 Wiley-Liss, Inc. SOMATOSTATIN ANALOG RC-160 INHIBITS THE GROWTH OF HUMAN OSTEOSARCOMAS IN NUDE MICE Jacek PIN SKI',^, Andrew V. SCHALLY~,~,~, Gabor HALMOS~,~, Karoly SZEPESHAZI',~ and Kate GROOT' 'Endocrine, Polypeptide and Cancer Institute, VeteransAffairs Medical Center, and 2Departmentof Medicine, Tulane University School of Medicine, New Orleans, LA 70146, USA. We investigated the effects of the potent somatostatin analog RC-160 on the growth of human osteosarcoma cell lines SKES- I and MNNG/HOS, transplanted into nude mice or cultured in vitro. Growth of SK-ES-I and MNNG/HOS tumors in nude mice was significantly inhibited after 4 weeks of treatment with daily S.C. injections of 100 pg RC-160, as measured by a reduction in tumor volume and weight. Histologically, the number of mitotic cells was decreased in the groups treated with RC- 160. In mice bearing either tumor model, administration of RC- I60 significantly decreased serum growth hormone and insulin-like growth factor I (IGF-I) levels. Specific highaffinity receptors for somatostatin and epidermal growth factor were found on membranes of MNNG/HOS tumors but not on SK-ES- I tumors. Receptor analyses also demonstrated highaffinity binding sites for ICF-I on membranes of both tumors. In cell cultures, the proliferationrate of MNNG/HOS cells, but not of SK-ES- I, was significantly suppressed by RC- 160. Our findings demonstrate that RC- I60 can significantly inhibit the growth of SK-ES-I and MNNG/HOS osteosarcomas in mice. o 1996 Wiley-Liss, Inc. @s-Tyr-D-Trp-Lys-Val-Cyk-Trp-NH2 (RC-160; Octastatin), have been synthesized in our laboratory and shown to inhibit the growth of experimental pancreatic, prostatic, colorectal, gastric and mammary carcinomas (Pinski et al., 1994; Schally, 1988). Somatostatin analogs also reduced local proliferation of experimental chondrosarcomas in vivo (Redding and Schally, 1983; Reubi, 1985). In this study, we have evaluated the effect of somatostatin analog RC-160 on the growth of the human osteosarcoma cell lines SK-ES-1 and MNNG/HOS in vivo and in vitro. The 2 cell lines, MNNG/HOS and SK-ES-1, were chosen because they are representative of malignant human bone tumors, well characterized and known to proliferate in vitro and to develop tumors in athymic nude mice (Bloom, 1972; Rhim et al., 1975). MATERIAL AND METHODS Peptides Somatostatin analog RC-160 (D-Phe-C$s-Tyr-D-Trp-Lys-Val-QhOsteogenic sarcomas represent the most common type of Trp-NH2) was made by classical synthesis by Novabiochem primary bone tumor in children and young adults (Rosen, (Laufelfingen, Switzerland) and supplied by Debiopharm (Lau1993). Surgery, radiation and chemotherapy are of limited sanne, Switzerland). For daily injections, RC-160 was diseffectiveness in the treatment of osteosarcomas, and other solved in 0.1% DMSO in saline solution. therapeutic approaches must be explored. Insulin-like growth Animals factor-I (IGF-I) is a potent stimulant of human and murine Male athymic (NCr nulnu) nude mice, approximately 6 osteosarcoma cell growth (Fournier et al., 1993; Kappel et al., 1994; Lopaczynski et al., 1993; Pirskanen et al., 1993; Pollak et weeks old on arrival, were obtained from NCI (Bethesda, MD) al., 1990, 1992; Raile et al., 1994; Scheven et al., 1991). Most of and maintained under pathogen-limited conditions. Their care the circulating IGF-I is produced in the liver, where transcrip- was in accord with institutional guidelines. tion of the IGF-I gene is positively regulated by growth Studies on tumor growth hormone (GH) (Pollak et al., 1990,1992). It has been demonIn the first experiment, xenografts were initiated by S.C. strated that the receptors for IGF-I are present on osteogenic injection of 1 x lo7SK-ES-1 cells into the right flanks of 5 male sarcomas (Fournier et at., 1993; Kappel et al., 1994; Lopaczyn- nude mice. Tumors resulting after 4 weeks were aseptically skietal., 1993; Pollaketal., 1990,1992; Raileetaf, 1994). It has dissected and mechanically minced; 3-mm3 pieces of tumor also been shown that proliferation of human and murine tissue were transplanted S.C. by trocar needle into 20 mice osteosarcomas in cell cultures i s enhanced by the addition of under methoxyflurane anesthesia. One week after transplantaIGF-I (Fournier et al., 1993; Kappel et al., 1994; Lopaczynski et tion, when tumors had grown to a volume of approx. 20 mm3, al., 1993; Pirskanen et al., 1993; Pollak et al., 1990, 1992; Raile mice were randomized and divided into 2 experimental groups et al., 1994; Scheven et al., 1994). GH can likewise exert a direct of 10 animals each, which received the following treatments for stimulatory effect on the growth of human osteosarcoma cells 4 weeks: (i) daily saline injections only, control, or (ii) RC-160, in vitro (Scheven et al., 1991). injected S.C. at a daily dose of 100 pg/day/animal. In view of these findings, the aim of a potential therapy In the second experiment, xenografts were initiated by S.C. could be to inhibit GH release from the pituitary to lower GH injection of 1 X lo7 MNNG/HOS cells into nude mice. and IGF-I levels. The benefit of this strategy is supported by Developed tumors were transplanted into 20 mice, and the the inhibitory effect of hypophysectomy on the metastatic experiment was carried out as described above. behavior of a murine osteosarcoma (Pollak et al., 1992). Tumors were measured with calipers, and tumor volume was Somatostatin analog RC-160 also inhibits metastatic behavior calculated as length X width x height x 0.5236, as described of RIFl osteosarcomas in mice (L. Wallace, personal commu- previously (Redding and Schally, 1983). At the end of the nication). treatment period, treated animals and control mice were Somatostatin and its analogs inhibit the release of G H as anesthetized with methoxyflurane and killed by decapitation. well as the secretion or action of growth factors such as IGF-I and epidermal growth factor (EGF) (Schally, 1988). Signifi3Towhom correspondence and requests for reprints should be sent, cant decreases in basal and arginine-stimulated serum G H and IGF-I levels were noted in patients with neoplasms potentially at VA Medical Center, 1601Perdido Street, New Orleans, LA 70146, dependent on IGF-I who received a somatostatin analog USA. Fax: (504) 566-1625. (Pollak et al., 1989). Superactive octapeptide analogs of somatostatin with a long duration of action, such as D-PheReceived: November 6,1995. 871 HORMONAL SUPPRESSIONOF OSTEOSARCOMAS Trunk blood was collected and centrifuged, and serum was frozen for hormone studies. Tumors were carefully cleaned and weighed, and samples were taken for histology and receptor studies. Histologicalprocedures A part of each tumor was fixed in 10% buffered formalin. Specimens were embedded in Paraplast (Oxford Labware, St. Louis, MO). Six-micrometer-thick sections were cut and stained with hematoxylin-eosin. Mitotic and apoptotic cells were counted in 10 standard high-power microscopic fields containing, on the average, 300 cells, and their numbers per 1,000 cells were accepted as the mitotic and apoptotic indices, respectively. Radioimmunoassays (RLA)of IGF-I and GH All serum samples for IGF-I determination were extracted by a modified acid-ethanol method described earlier (Breier et al., 1991). Serum IGF-I levels were determined using antiIGF-I anti-serum (UB3-189) provided by the National Hormone and Pituitary Program of the National Institute of Diabetes and Digestive and Kidney Diseases at the final dilution of 1:14,000. IGF-I (a gift from Genentech, South San Francisco, CA) was iodinated by the Chloramine-T method. IGF-I standards in the range of 8-2,000 pg/tube were calibrated against WHO 1st International Reference Reagent 1988, IGF-I 87/518, supplied by the Nichols Institute (San Juan Capistrano, CA). Serum GH levels were determined by double-antibody RIA using materials supplied by NIDDK. Inter-assay and intraassay coefficients of variation were less than 15% and lo%, respectively. Receptor assay Receptors for somatostatin, EGF and IGF-I on the membranes of SK-ES-1 and MNNG/HOS tumors were measured as previously described (Srkalovic et al., 1989; Szepeshazi et al., 1992). Characteristics of somatostatin receptors were determined in competitive binding experiments using the radioligand RC-160as described previously (O’Byrne et al., 1994). The LIGAND PC computerized curve-fitting program of Munson and Rodbard (1980) was used to determine the types of receptor binding, dissociation constant (&) values and the maximal binding capacity (Bmaw) of receptors. In vitro studies The human osteosarcoma cell lines SK-ES-1 and MNNG/ HOS were obtained from the ATCC (Rockville, MD). SKES-1 was grown as a monolayer in McCoy’s 5a medium supplemented with 15% FBS, antibiotics and antimycotics. MNNG/HOS was grown as a monolayer in MEM supplemented with 10% FBS, antibiotics and antimycotics. Cultures were incubated at 5% C 0 2 in air at 37°C. Cells from 7040% confluent cultures were seeded into Costar (Cambridge, MA) ’24-well plates at a density of 5 x lo3cells/well. After 48 hr (day 0), media were replaced with fresh media containing RC-160 in concentrations of 10-11-10-6 M. The cell number for each well was determined in a Coulter (Hialeah, FL) counter after detachment of cells by trypsinization on day 4. Cell viability, measured by Trypan blue exclusion, was more than 90%. Statistical methods Statistical analyses of the data were performed using Duncan’s new multiple range test (Steel and Torrie, 1976) and Student’s 2-tailed t test. All p values are based on 2-sided hypothesis testing. RESULTS Effect of RC-160 on growth of osteosarcomas in nude mice There were no significant differences in body weights between treated and control groups at the end of the experi- 1 Iooo A -0- T Control 800 m - -,,, 600 28! 400 E E I z 2 200 0 0 2 1 3 4 3 4 WEEKS OF TREATMENT 0 1 2 WEEKS OF TREATMENT FIGURE1 - Tumor volumes in athymic nude mice bearing S.C. transplanted (a) SK-ES-1 and (b) MNNG/HOS human osteosarcomas during treatment with somatostatin analog RC-160 administrated by daily S.C. injections at a dose of 100 kg/animal/day. Treatment was started when tumors measured approximately 16-22 mm3 and lasted for 4 weeks. Vertical bars represent S.E.; *p < 0.05, **p < 0.01 vs. control. ments. In experiment I, after 3 weeks of treatment, the tumor volume of SK-ES-1 osteosarcomas in the group receiving RC-160 was significantly ( p < 0.05) reduced to 134.1 k 54.0 mm3 as compared with the control group (479.6 f 121.4 mm3), corresponding to a 72% decrease in tumor volume (Fig. la). The final tumor volume and tumor weight were significantly ( p < 0.01) diminished in animals treated for 4 weeks with RC-160 to 210.3 f 71.7 mm3 and 0.25 f 0.1 g, respectively, compared with those for controls (764.0 f 167.3 mm3 and 0.78 2 0.1 g) (Table I). In experiment 11, RC-160 therapy similarly inhibited the growth of MNNG/HOS tumors (Fig. lb). The mean tumor volume and weight were significantly ( p < 0.05) reduced in animals receiving RC-160 for 4 weeks to 455.3 f 71.8 mm3and PINSKI ETAL. 872 TABLE I - EFFECT O F TREATMENT FOR 4 WEEKS WITH SOMATOSTATIN ANALOG RC-160 ON BODY AND TUMOR WEIGHT AND FINAL TUMOR VOLUME IN NUDE MICE BEARING XENOGRAFTS O F THE HUMAN OSTEOSARCOMA CELL LINES SK-ES-1 AND MNNGIHOS Tumor volume (mm') Treatment group Experiment I (SK-ES-1) Control RC-160 Experiment I1 (MNNGiHOS) Control RC-160 Body weight (g) Tumor weight (g) 764.0 t 167.3 210.3 t 71.72 25.2 f 1.0 24.6 f 0.9 0.78 f 0.1 0.25 t 0.1' 925.2 t 66.0 455.3 f 71.8* 24.8 t 0.8 23.5 f 0.5 1.0 t 0.2 0.48 0.06l Initial Final 22.6 t 3.6 21.3 t 3.8 16.3 t 1.5 20.0 f 1.7 * Values are means + S.E.-lp < 0.05, ' p < 0.01 vs. control. TABLE I1 - SERUM GROWTH HORMONE (GHI AND INSULIN-LIKE GROWTH FACTOR I (IGF-I) LEVELS IN NUDE MICE WITH XENOGRAFTS O F THE HUMAN OSTEOSARCOMA CELL LINES SK-ES-1 AND MNNGIHOS AFTER TREATMENT WITH SOMATOSTATIN ANALOG RC-160 ~~ ~~~~ Treatment group Exoeriment I (SK-ES-11 ' kontrol RC-160 Experiment 11 (MNNGIHOS) Control RC-160 GH (nglml) IGF-I (ngiml) 12.0 k 1.8 3.6 t 0.5* 374.6 t 81.1 95.4 t 12.8l 8.8 f 2.3 4.5 + 0.4l 316.6 f 45.1 185.2 f 10.1' Values are means +- .S.E..Jp < 0.05, ' p < 0.01 vs. control. 0.48 f 0.06 g compared with those in the control group, which were 925.2 2 66.0 mm3 and 1.0 r?T 0.2 g, respectively (Table I). Histological findings Histologically, SK-ES-1 tumors were composed of undifferentiated, large polygonal cells arranged close to each other. Cells had narrow cytoplasms and round, large nuclei with prominent nucleoli. Necrotic foci could be found in all tumors. The mitotic index value showed a slight and statistically not significant decrease in tumors treated with RC-160 (4.8 t 1.0 vs. 7.8 f 1.4 in controls). The ratio of apoptotic to mitotic indices was significantly ( p < 0.01) higher in SK-ES-1 tumors receiving RC-160 (4.36 t 2.6 vs. 1.9 r?T 0.31 in controls). MNNG/HOS tumors were highly cellular. Tumor cells were arranged without any special orientation. Cytoplasms were slightly basophilic. Nuclei of tumor cells were large, round, elongated in some areas and contained prominent nucleoli. No signs of differentation or osteoid formation could be observed. The mitotic index value decreased significantly ( p < 0.05) to 14.0 2 2.0 in tumors treated with RC-160 as compared with the control group (22.5 t 3.7). The ratio of apoptotic to mitotic indices in MNNG/HOS tumors was significantly ( p < 0.05) increased by treatment with RC-160 at the end of the experiment (0.81 t 0.15 vs. 0.36 +- 0.07 in controls). Serum hormone levels Serum G H and IGF-I levels in control nude mice and in animals treated with RC-160 in both experiments are shown in Table 11. Therapy with RC-160 significantly reduced serum GH levels to 3.6 0.5 ngiml in experiment I ( p < 0.01) and to 4.5 t 0.4 ngiml in experiment I1 ( p < 0.05), as compared with controls (12.0 2 1.8 ngiml and 8.8 f 2.3 ngiml, respectively). Treatment with RC-160 also significantly ( p < 0.05) decreased serum IGF-I concentrations in animals bearing SKES-1 tumors, to 95.4 12.8 ngiml, and in mice with MNNGi HOS tumors, to 185.2 ? 10.1, as compared with controls (374.6 2 81.1 ngiml and 316.6 f 45.1 ngiml, respectively) (Table 11). * Receptor findings The binding characteristics of receptors for somatostatin, IGF-I and EGF in SK-ES-1 and MNNGiHOS tumors were analyzed following treatment with somatostatin agonist RC160, and the results are presented in Table 111. Receptor assays on SK-ES-1 tumor membranes showed high-affinity binding sites for IGF-I (&: 0.7 2 0.01 nM), but the receptors for somatostatin and E G F were absent (Table 111). The concentration of IGF-I receptors was significantly ( p < 0.01) increased by treatment with RC-160 to 249.5 -C 4.9 fmolimg protein, as compared with the control group (154.2 ? 6.1 fmolimg protein), while the affinity of receptors was not changed (& = 0.7 t 0.01 nM). High-affinity binding sites for IGF-I were also found on MNNGiHOS control tumors (& = 0.48 f 0.08 nM) and RC-160-treated tumors (& = 0.76 t 0.17 nM). A significant ( p < 0.05) increase in binding capacity (Bma)of IGF-I receptors was again observed after treatment with RC-160, from 74.9 2 5.9 fmolimg protein found in the control tumors to 113.3 f 20.3 fmol/mg protein. Receptor assays on membranes of MNNGiHOS tumors also demonstrated highaffinity binding sites for somatostatin (& = 9.52 ? 0.05 nM) and E G F (& = 0.88 f. 0.08 nM). Therapy with RC-160 significantly ( p < 0.01) increased the binding capacity of receptors for somatostatin on membranes of MNNG/HOS tumors by about 85% (Table 111). A significant ( p < 0.05) reduction in binding capacity (Bmax)of E G F receptors was observed after treatment with RC-160, from 251.1 ? 10.3 fmolimg protein found in control tumors to 85.0 2 3.37 fmolimg protein in tumors treated with RC-160 (Table 111). Effect of RC-160 on cellularproliferation The proliferation of MNNGiHOS cells was significantly ( p < 0.01) inhibited by RC-160 at 10-*o-10-6 M concentrations (Fig. 2). At concentrations of lo7 and lo6 M, RC-160 decreased the proliferation of MNNGiHOS cells by 30.4% and 43.6%, respectively, as compared with controls (Fig. 2). Proliferation of SK-ES-1 cells was not affected by RC-160 at concentrations of 10-11-10-6 M (not shown). DISCUSSION Our findings demonstrate that somatostatin agonist RC-160 can significantly inhibit growth of the xenografts of both osteosarcoma cell lines in vivo. Anti-neoplastic actions of somatostatin agonists may involve multiple mechanisms. A significant fall in G H levels induced by RC-160 could, through mechanisms involving suppression of endogenous growth factors, particularly IGF-I, be of major importance for the inhibition of tumor growth. IGF-I has been shown to play an important role in growth of osteosarcomas (Fournier et al., 1993; Kappel et al., 1994; Lopaczynski et al., 1993; Pirskanen et al., 1993; Pollak et al., 1990, 1992; Raile et al., 1994; Scheven et al., 1991). Stimulatory effects of IGF-I on the proliferation of various human and murine osteosarcoma cell lines have been clearly demonstrated (Fournier et al., 1993; Kappel et al., 1994; Lopaczynski et al., 1993; Pollak et al., 1990, 1992; Raile et al., 1994). The growth of some osteosarcoma cell lines could be inhibited in vitro by monoclonal antibodies and anti-sense HORMONAL SUPPRESSION OF OSTEOSARCOMAS 873 TABLE I11 - BINDING CHARACTERISTICS OF SOMATOSTATIN,EGF AND IGF-I RECEPTORS IN MEMBRANES OF SK-ES-1AND MNNGIHOS TUMORS AFTER IN VIVO TREATMENT WITH SOMATOSTATINANALOG RC-160 Somatostatin EGF IGF-I Experiment I (SK-ES-1) Control ND ND ND ND 0.7 f 0.01 RC-160 ND ND ND ND 0.78 f 0.09 Experiment I1 (MNNG/HOS) Control 9.52 f 0.05 699.1 f 13.4 0.88 f 0.08 251.1 f 10.3 0.48 2 0.08 RC-160 13.1 f 0.05 1293.1 f 160.22 0.55 f 0.03l 85.0 f 3.372 0.76 +- 0.17 154.2 2 6.1 249.5 f 4.92 74.9 f 5.9 113.3 & 20.3' < 0.01 vs. control. N.D., not detectable. Binding Values are means 2 S.E.-lp < 0.05, characteristics were obtained from 10-point displacement experiments in triplicate tubes. 3500 1 * * * * * U 3z W 0 Y CONTROL -11 -10 -9 -8 -7 -6 LOG CONCENTRATION (M) FIGURE 2 - Anti-proliferative effect of somatostatin analog RC160 on MNNG/HOS cells in vitro as measured by cell number. Cells were cultured in the presence of RC-160 in concentrationsof lo-" to M. Vertical bars represent S.E.; *p < 0.01 vs. control. oligonucleotides to the IGF-I receptor (Kappel et al., 1994; Pollak et al., 1990; Raile et al., 1994). Our finding of high-affinity binding sites for IGF-I in membranes of MNNG/HOS and SK-ES-1 tumors is in agreement with previous reports by other groups on the existence of high-affinity receptors for IGF-I in various human and murine osteogenic sarcoma cell lines (Fournier et a/., 1993; Kappel et al., 1994; Lopaczynski et a/., 1993; Pollak et a/., 1990, 1992; Raile et al., 1994). IGF-I and IGF-I1 bind with different affinities to the type-I IGF receptor, which is thought to mediate the biological effects of both ligands through tyrosine kinase-type activity (Fournier et al., 1993). We observed that chronic treatment of nude mice with RC-160 increased the concentration of IGF-I receptors of both osteosarcoma tumors. This might reflect a compensatory process caused by suppression of the endocrine or local production of IGF-I induced by the somatostatin analog. RC-160 might also inhibit growth of osteosarcomas through the suppression of the release of GH itself. GH was found to stimulate proliferation of the human osteogenic sarcoma cell line SaOS-2 in vitro (Scheven et al., 1991), and these effects were abolished by an anti-IGF antibody, suggesting that G H acted by increasing the autocrine production of IGFs (Scheven et al., 1991). Since high-affinity receptors for somatostatin are present on MNNG/HOS tumor membranes, somatostatin agonist RC160 could also directly inhibit the growth of MNNG/HOS cells. In our study, the proliferation of MNNG/HOS cells in vitro, but not that of somatostatin receptor-negative SK-ES-1 cells, was suppressed by RC-160. Somatostatin and its analogs stimulate tyrosine phosphatase and promote the dephosphorylati'on of EGF receptors (Buscail et al., 1994; Liebow et al., 1989). We have shown that somatostatin agonist RC-160 produced a great reduction in the concentration of EGF receptors on membranes of MNNG/HOS tumors. Thus, the ability of MNNGiHOS tumor cells to respond to transforming growth factor (Y and EGF could have been decreased in our study. This down-regulation of E G F receptors in MNNG/ HOS osteosarcomas after therapy with RC-160 is in agreement with similar findings in other tumors (Liebow et al., 1989; Pinski et at., 1994; Schally, 1988). The inhibitory effect of RC-160 on the growth of SK-ES-1 tumors in vivo observed in our study is probably mainly due to suppression of G H and IGF-I secretion since we did not find somatostatin receptors on membranes of this tumor. However, the fact that our assay did not detect somatostatin receptors on membranes of SKES-1 tumors may also be explained by the existence of several somatostatin receptor subtypes (Bell and Reisine, 1993; Buscail et al., 1994). Molecular cloning revealed the presence of 5 structurally related integral membrane glycoproteins, which are pharmacologically distinct high-affinity somatostatin receptors with different regional distributions and functions (Bell and Reisine, 1993; Buscail et al., 1994). Pharmacological studies on the characteristics of somatostatin receptors showed that the different subtypes exhibit major differences in their affinities for structural analogs of somatostatin (Bell and Reisine, 1993; Buscail et al., 1994). In our study, 1251-labeled RC-160, which is more selective for SSTR-2 and SSTR-5 subtypes (Buscail et al., 1994), was used as a tracer in radioreceptor assays. Thus, the fact that we found somatostatin receptors on MNNG/HOS osteosarcomas and failed to detect them on SK-ES-1 tumors may be due to different binding affinities of RC-160 to various subtypes of somatostatin receptor. However, our receptor results indicate a significant increase in the concentration of binding sites for somatostatin on membranes of MNNG/HOS tumors treated with the somatostatin analog. The finding that 4 weeks of administration of RC-160 did not produce a down-regulation of somatostatin receptors indicates that this therapy could be applied for prolonged periods of time. This lack of down-regulation of somatostatin receptors after therapy with RC-160 is also similar to findings observed in other tumors (Srkalovic et aL, 1989; Pinski et al., 1994). 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