Introduction

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Growth factors and their receptors are key regulators of cell growth and survival. Growth factor involvement in carcinogenesis is common and includes overexpression of growth factor receptors (Baselga and Mendelsohn, 1994), enhanced kinase activity (Resnik et al., 1998), or stimulation of cancer cell growth by autocrine/paracrine mechanisms (Yee et al., 1989).

It has been postulated that cells require constant activation of survival pathways; among a variety of growth factors, insulin-like growth factor-1 (IGF-1) is the most effective protector against programmed cell death (apoptosis) induced by c-myc (Harrington et al., 1994). The antiapoptotic effects of IGF-1 have been reported to be more pronounced in vivo than in vitro (Resnicoff et al., 1995a,b). High levels of circulating IGF-1 have been found in prostate (Chan et al., 1998) and breast cancer patients (Peyrat et al., 1993). IGF-1 has been shown to protect cells against a variety of noxious stimuli, including cytokine withdrawal (Rodriguez-Tarduchy et al., 1992), overexpression of interleukin-1 -converting enzyme (Jung et al., 1996), or clinically used anticancer agents (Sell et al., 1995; Dunn et al., 1997). The mechanism by which IGF-1 and its receptor protect cells from apoptosis involves elements of the mitogen-activated protein kinase and phosphoinositide 3-OH kinase [PI(3)K] pathways (Parrizas et al., 1997). Recent studies have identified a linear survival cascade consisting of IGF-1, the IGF-1 receptor, PI(3)K, and the serine/threonine kinase Akt (also termed protein kinase B or PKB) (Kulik and Weber, 1997). Activated Akt phosphorylates Bad, a proapoptotic member of the Bcl-2 family, thereby abolishing Bad's apoptotic properties (Datta et al., 1997; del Peso et al., 1997). An IGF-1 dependent, but PI(3)K and Akt-independent survival pathway also has been described (Kulik and Weber, 1998). The role of the IGF-1 receptor in cellular transformation has been studied extensively (Sell et al., 1994; Baserga, 1995), and controlling IGF-1 receptor function has become a target for anticancer therapy (Baserga, 1996). Despite the large body of evidence linking IGF-1 and its receptor to the establishment of the transformed phenotype, however, therapies based on specific inhibition of IGF-1 receptor function are just beginning to emerge. Although several small molecules inhibiting various downstream targets of the IGF-1 receptor have been identified, few agents have been described that selectively inhibit IGF-1 receptor signaling.

Recent work from our laboratory has identified a novel antiproliferative agent, SC-9 {4-(benzyl-(2-[(2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylamino butyric acid}, from a combinatorial library modeled after natural product phosphatase inhibitors (Rice et al., 1997; Wipf et al., 1997; Vogt et al., 1998). Studies of SC-9 in cultured mouse embryonic fibroblasts transformed with the simian virus large T antigen (SV40 MEF) showed selective inhibition of transformed cell growth and suggested that SC-9 affected elements of IGF-1 signaling (Vogt et al., 1998). Because these studies involved long-term (48 h) exposure to the drug, however, the functional significance of these observations remains unclear. In addition, we were unable to unambiguously determine whether SC-9 induced programmed cell death in these cells.

In the current study, we have extended our previous observations by investigating the induction of apoptosis by SC-9 in 32D mouse myeloid progenitor cells, a well studied and convenient model for programmed cell death (Nunez et al., 1990). Not only did we demonstrate that SC-9 caused apoptosis but also in contrast to interleukin-3 (IL-3) withdrawal or treatment with the clinically used antineoplastic agent etoposide, SC-9-induced apoptosis was resistant to both an overabundance of IGF-1 and to overexpression of the IGF-1 receptor. Furthermore, although exogenous IGF-1 was able to maintain or elevate levels of Cdc2, a biochemical indicator of a functional IGF-1 receptor pathway, in IL-3-deprived and etoposide-treated cells, exogenous IGF-1 was unable to maintain Cdc2 expression after SC-9 treatment. Finally, we demonstrated in vivo antitumor activity of SC-9 against a murine small cell squamous carcinoma that is positive for the IGF-1 receptor. Taken together, our data suggest SC-9 has potential for the treatment of IGF-1-dependent tumors.

	Materials and Methods

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Chemical Compounds. The synthesis of compounds SC-9 and 4-{(2-[(5-methyl-2-phenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl}-2-decanoylamino butyric acid (SC-109) has been previously described (Rice et al., 1997; Wipf et al., 1997; Vogt et al., 1998). Compounds were isolated as racemic mixtures by chromatography on SiO₂ and characterized by NMR and high-resolution mass spectrometry.

Cell Culture. Four murine cell lines were used to probe the pharmacological effects of SC-9. All cells were grown in a humidified atmosphere of 5% CO₂ at 37°C. 32D/neo and 32D/Bcl-2 (clone 23) cells were a kind gift from Dr. Daniel Johnson (University of Pittsburgh Cancer Institute). The IGF-1 receptor overexpressing line 32D/GR15 and the empty vector control 32D/mscv were a kind gift from Dr. Renato Baserga (Kimmel Cancer Center, Philadelphia, PA). These cells were established from 32D cells (clone 3) by transfection with wild-type human IGF-1 receptor cDNA subcloned into the mscv retroviral vector (Romano et al., 1999; Valentinis et al., 1999). All 32D subclones were maintained as previously described (Fabisiak et al., 1997) in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, 2 mM glutamine, 1.25 µg/ml fungizone, and 10% WEHI-3B conditioned medium as a source of IL-3, an essential survival factor for 32D cells. LISN C4 cells, an NIH 3T3 cell line overexpressing human IGF-1 receptors (Kaleko et al., 1990) were obtained from Dr. Daniel Altschuler (University of Pittsburgh) and cultured in Dulbecco's modified Eagle's medium supplemented with 5% bovine calf serum as described (Altschuler et al., 1994). Murine SCCVII/SF squamous cell carcinoma tumors were produced by s.c. inoculation of 5 × 10⁵ exponential growing tumor cells from culture in the right flank of 6- to 10-week-old female C3H/HeJ mice. SCCVII/SF cells were generated from these tumors by routine dissection and resuspension in Dulbecco's minimum essential medium containing 20% FBS supplemented with 1% penicillin/streptomycin (Johnson et al., 1993).

Antiproliferative and Antitumor Activity of SC-9. The in vitro antiproliferative activity of SC-9 was determined by our previously described 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay with 48 h of continuous drug exposure (Vogt et al., 1998). The in vivo effect of SC-9 was evaluated and compared with other clinically used anticancer agents with the in vivo excision clonogenic cell survival assay (Johnson et al., 1993). Briefly, animals (three to four mice per treatment group) with established tumors (day 14 postimplant) were treated with a single i.p. dose of either vehicle (Cremophor EL/ethanol), SC-9, cis-diamminedichloroplatinum (cisplatin), or cis-diammine(1,1-cyclobutanedicarboxylato)platinum (carboplatin) at varying doses. After 24 h, tumors were harvested and single cell preparations generated as previously described (Johnson et al., 1993). Viable tumor cells as determined by trypan blue exclusion were plated at various dilutions and after 7 days of incubation, colonies were counted and numbers of clonogenic cells per gram of tumor were enumerated. The surviving fraction per gram of tumor was defined as the number of clonogenic tumor cells per gram of treated tumor divided by the number of clonogenic tumor cells per gram of control (untreated) tumor.

Nuclear Morphology. Exponentially growing 32D cells were treated with drug or vehicle for 24 h and collected by centrifugation (400g, 2 min). Cells were washed in PBS, fixed with 2% paraformaldehyde in PBS, and stained with Hoechst 33342 fluorescent dye (1 µg/ml) as described (Fabisiak et al., 1997). For quantitation of apoptosis, at least 300 cells were examined by fluorescence microscopy. Apoptotic cells were identified by condensed, bright-staining nuclei, usually very rounded, and sometimes fragmented into distinct sections.

Western Blotting. 32D cells were seeded at 1.5 × 10⁵ cells/ml and treated 2 days later with drug or vehicle for various periods of time. Cell pellets were treated with lysis buffer (30 mM HEPES, pH 7.5, 1% Triton X-100, 10% glycerol, 5 mM MgCl₂, 25 mM NaF, 1 mM EGTA, 10 mM NaCl, 2 mM Na₃VO₄, 10 µg/ml trypsin inhibitor, 10 µg/ml aprotinin, 25 µg/ml leupeptin, 2 mM PMSF, and 6.4 mg/ml Sigma-104 phosphatase substrate). Cleared lysates were electrophoresed on 4 to 20% gradient gels (NOVEX, San Diego, CA), transferred to nitrocellulose, and immunoblotted with antibodies against Cdc2 (17) or the IGF-1 receptor  subunit (C-20; both from Santa Cruz Biotechnology, Santa Cruz, CA). Positive antibody reactions were visualized with peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and an enhanced chemiluminescence detection system (Renaissance; NEN, Boston, MA) according to manufacturer's instructions. For quantitation of protein expression levels, X-ray films were scanned on a Molecular Dynamics personal SI densitometer and analyzed with the ImageQuant software package (version 4.1; Molecular Dynamics, Sunnyvale, CA). Band intensities were normalized to untreated control samples harvested at the same time.

IGF-1 Protection Experiments. As described above, 32D cells were treated with vehicle, SC-9, or etoposide. For IL-3 withdrawal experiments, the cells were centrifuged for 5 min at 400g, washed five times in cold PBS, and resuspended in medium containing 10% FBS. Exogenous human recombinant IGF-1 (Sigma Chemical Co., St Louis, MO) was added to drug treated or IL-3-deprived cells at the time of treatment. The percentage of cells with apoptotic nuclei was determined after 24 h as described above.

IGF-1 Receptor Tyrosine Phosphorylation Assay. For receptor autophosphorylation experiments, LISN C4 cells were grown to near confluency and growth arrested in 1% fetal calf serum for 24 h. SC-9 or vehicle was included during the last 4 h of starvation, and cells were stimulated with IGF-1 (50 ng/ml) for 15 min. Monolayers were rinsed once with PBS and lysed in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 10 mM sodium pyrophosphate, 0.2 mM Na₃VO₄, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 10 µg/ml aprotinin. Lysates were cleared by centrifugation for 2 min at 3000g. Then 250 to 500 µg of protein lysate were brought up to 1 or 2 volumes with washing buffer [20 mM HEPES, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 10% glycerol, 0.2 mM Na₃VO₄, 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 2 µg/ml aprotinin] and immunoprecipitated overnight at 4°C with 1 µg of anti-IGF-1 receptor -subunit antibody (IR-3, AB-1; Oncogene, Cambridge, MA) along with whole mouse IgG conjugated to agarose (Sigma Chemical Co.) as a carrier. Immunoprecipitates were collected by centrifugation at 13,000g for 4 min., washed three times with washing buffer, and boiled in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer. Supernatant proteins (25 µl) were separated on 4 to 12% Tris-glycine gradient gels and immunoblotted with a horseradish peroxidase-conjugated anti-phosphotyrosine antibody (PY20-HRP; Transduction Laboratories, Lexington, KY). Duplicate gels were blotted with a rabbit polyclonal anti-IGF-1 receptor -subunit antibody (C-20; Santa Cruz Biotechnology) followed by a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) to confirm the presence of the receptor.

	Results

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SC-9-Induced Apoptosis in 32D/neo But Not 32D/Bcl-2 Cells. The antiproliferative activity of SC-9 against human MDA-MB-231 breast cancer and mouse embryonic cells has been reported previously (Wipf et al., 1997; Vogt et al., 1998). To more precisely probe mechanisms of SC-9 antiproliferative activity, we used 32D cells as a convenient and well established model of programmed cell death (apoptosis) (Nunez et al., 1990). Cells were treated continuously with SC-9 for 24 h and three different parameters indicative of apoptosis were measured. With Hoechst fluorescent dye staining, we found that SC-9 caused concentration-dependent nuclear morphology changes characteristic of apoptosis in 32D cells (Figs. 1 and 2) with an IC₅₀ value of 15 µM. Apoptotic death was further confirmed by analysis of cellular DNA by agarose gel electrophoresis and by flow cytometry. Cells treated with SC-9 also displayed internucleosomal DNA fragments ("ladders") as well as a population of cells with <2 N DNA content (data not shown). In contrast, SC-109, a congeneric analog of SC-9, which we previously described and that differs from SC-9 only in the lack of a phenyl and benzyl moiety (Vogt et al., 1998), did not induce changes in nuclear morphology or degradation of DNA into internucleosomal fragments (Fig. 2 and data not shown). Overexpression of the antiapoptosis gene product Bcl-2 protected 32D cells against SC-9-induced apoptosis (Figs. 1 and 2), indicating that SC-9 specifically affected signaling mechanisms leading to programmed cell death.

View larger version (115K): [in this window] [in a new window]   Fig. 1.   SC-9 induces apoptosis in 32D/neo but not 32D/Bcl-2 cells. 32D/neo (A, B) and 32D/Bcl-2 (C, D) cells were exposed to vehicle (A, C) or 30 µM SC-9 (B, D) for 24 h, centrifuged, fixed, stained with Hoechst 33342 fluorescent dye, and photographed. Arrows denote apoptotic nuclei that show chromatin condensation and bright micronuclei. Bar = 3 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   A closely related analog of SC-9 did not alter nuclear morphology and Bcl-2 expression blocked apoptosis induced by SC-9. 32D/neo and 32D/Bcl-2 cells were treated with the indicated concentrations of either SC-9 or SC-109 for 24 h, and stained with Hoechst 33342 fluorescent dye. Approximately 300 nuclei were scored for apoptotic morphology as described in the legend to Fig. 1. Apoptosis is expressed as the percentage of apoptotic cells in the entire cell population.

IGF-1 Protects against IL-3 Withdrawal and Etoposide But Not against SC-9. To investigate whether IGF-1 survival pathways were functionally involved in the apoptotic properties of SC-9, we first assessed whether SC-9-induced apoptosis could be overcome by high concentrations of IGF-1. To demonstrate that these cells, which lack insulin receptor substrate-1 and insulin receptor substrate-2 (Wang et al., 1993; Myers et al., 1996) responded to IGF-1 with increased cell survival, we treated IL-3-deprived cells with IGF-1. Withdrawal of IL-3 resulted in 57% apoptosis after 24 h and IGF-1 (100 ng/ml) reduced apoptosis in cytokine-deprived cells by 40% (Fig. 3). Cells were then treated with SC-9 in the presence or absence of exogenous IGF-1 for 24 h and analyzed for apoptotic morphology. In contrast to IL-3 withdrawal, addition of IGF-1 did not significantly reduce apoptosis by SC-9 (Fig. 3). IGF-1 did, however, cause a statistically significant decrease in the apoptosis induced by the clinically used antineoplastic agent etoposide, consistent with previous reports (Sell et al., 1995).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   IGF-1 protects against IL-3 withdrawal and etoposide but not against SC-9. 32D/neo cells were treated with SC-9 (10 or 30 µM), etoposide (1 µM), or were deprived of IL-3 for 24 h in the presence or absence of exogenously added IGF-1 (100 ng/ml). Data are means ± S.E. from the indicated numbers of independent experiments. Statistical analysis was by a two-tailed Student's t test assuming unequal variances compared with samples that did not receive IGF-1. *P < .05, **P < .01.

IGF-1 Prevents Down-Regulation of Cdc2 in Response to IL-3 Withdrawal, Partially after Etoposide, But Not after SC-9. We next asked whether exogenous IGF-1 was able to maintain Cdc2 levels, a biochemical indicator of a functional IGF-1 receptor pathway, during SC-9 treatment. 32D/neo cells were treated with SC-9 or etoposide, or deprived of IL-3 for 24 h in the presence or absence of 100 ng/ml IGF-1. Western blotting of cellular lysates with an anti-Cdc2 antibody showed that IGF-1 prevented down-regulation of Cdc2 by cytokine withdrawal, but not by SC-9 (Fig. 4, top). An equitoxic concentration of etoposide (1 µM) did not appreciably decrease Cdc2. In contrast to SC-9, addition of IGF-1 to etoposide-treated cells resulted in elevated Cdc2 expression. Because there is no known involvement of etoposide in IGF-1 receptor signaling, our data support the hypothesis that SC-9 counteracted the ability of IGF-1 to maintain Cdc2 levels, presumably by affecting a target in the IGF-1 receptor pathway. Immunoblot analysis with an anti-IGF-1 receptor -subunit antibody confirmed the presence of the IGF-1 receptor in all cases, although a significant loss of receptor occurred with 30 µM SC-9 and, to a lesser extent, with 1 µM etoposide (Fig. 4, middle).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   IGF-1 prevents down-regulation of Cdc2 by IL-3 withdrawal, but not by SC-9. Cells were deprived of IL-3 (-IL-3) or treated with 1 µM etoposide, or 10 µM and 30 µM SC-9 in the presence or absence of exogenous IGF-1 (100 ng/ml). After 24 h, lysates were separated on SDS-PAGE and immunoblotted with anti-Cdc2 (top) or anti-IGF-1 receptor (middle) antibodies. Identical results were obtained in a second independent experiment. GAPDH, loading control.

IGF-1 Receptor Overexpression Did Not Protect Cells against SC-9. Having demonstrated the inability of IGF-1 to rescue cells from apoptosis by SC-9, we next asked whether protection from SC-9 could be conferred by overexpression of the IGF-1 receptor. Three 32D subclones were examined for IGF-1 receptor expression by Western blot analysis and subjected to treatment with SC-9 for 24 h. As shown in Fig. 5A, 32D/neo cells had detectable IGF-1 receptor levels, whereas the 32D/mscv, which is a clonally derived cell line used as a control for the 32D/GR-15 cells, had very low IGF-1 receptor levels. As expected, the transfected 32D/GR-15 cells had the highest growth factor receptor levels. Figure 5B shows that SC-9 was equally active in three 32D cell lines despite the considerable difference in IGF-1 receptor levels Thus, we concluded that the apoptotic activity of SC-9 was independent of IGF-1 receptor density, which is consistent with a downstream target that is limiting in the pathway.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   IGF-1 receptor overexpression does not protect against SC-9-induced apoptosis. A, three subclones of 32D cells (neo, mscv, GR-15) were analyzed for expression of the IGF-1 receptor by Western blotting with an anti-IGF-1 receptor -subunit antibody. B, cells were treated with vehicle or SC-9 (30 µM) for 24 h and apoptotic nuclei were scored as described in Materials and Methods. Data are the average from two independent experiments ± range.

Kinetics of SC-9-Induced Cdc2 and IGF-1 Receptor Down-Regulation in 32D Cells. Having demonstrated an involvement of IGF-1 survival mechanisms in SC-9-induced apoptosis, we next probed potential sites of action for SC-9 in the IGF-1 receptor pathway. First, to exclude the possibility that the effects of SC-9 on Cdc2 and the IGF-1 receptor were a result of cell death rather than a direct consequence of compound treatment, we examined the temporal relationships between apoptosis, Cdc2 levels, and IGF-1 receptor levels in 32D cells after exposure to SC-9. 32D/neo and 32D/Bcl-2 cells were treated with SC-9 (30 µM), harvested at various times, and assayed for nuclear morphology and expression of Cdc2 and IGF-1 receptor as described in Materials and Methods. A 12- to 24-h exposure to SC-9 was required to reduce protein levels of Cdc2 in both cell lines (Figs. 6 and 7B). In contrast to Cdc2, however, loss of IGF-1 receptor was more pronounced in the 32D/neo cells and occurred earlier than in the Bcl-2 transfectants (Figs. 6 and 7C). Almost 50% of the IGF-1 receptor remained in Bcl-2-expressing cells 24 h after a 30-µM treatment with SC-9, whereas only 15% remained in the 32D/neo cells. Nonspecific toxicity, presumably due to growth and survival factor deprivation, was observed in both cell lines after 2 days in culture and resulted in a loss of Cdc2 and IGF-1 receptor even in the absence of SC-9 (Fig. 6). In 32D/neo cells, apoptosis showed some correlation with a loss of Cdc2, whereas in 32D/Bcl-2 cells loss of Cdc2 clearly preceded apoptosis (Fig. 7, A and B). For example, 32D/Bcl-2 cells treated with SC-9 showed a near complete loss of Cdc2 at 24 h, a time when only 10% of cells were undergoing apoptosis (Fig. 7, A and B). After 12 h, when loss of Cdc2 became first apparent (Fig. 7B), IGF-1 receptor levels were essentially unchanged in both cell lines (Fig. 7C). These data suggest that reduction of Cdc2 levels was likely to be a direct effect of SC-9, whereas loss of IGF-1 receptor may be a secondary effect.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effects of SC-9 on Cdc2 and IGF-1 receptor levels in 32D/neo and 32D/Bcl-2 cells. Exponentially growing 32D/neo (A) or 32D/Bcl-2 (B) cells were exposed to 30 µM SC-9 or vehicle. At the indicated time points, cells were collected by centrifugation, lysed, and lysates separated on SDS-PAGE. Western blot analysis was performed with antibodies against Cdc2 and the IGF-1 receptor -subunit. Equal protein loading was confirmed by immunoblotting the identical lysates with an anti-p38 mitogen-activated protein kinase antibody.

View larger version (28K): [in this window] [in a new window]   Fig. 7.   Cdc2 down-regulation precedes apoptosis and loss of IGF-1 receptor. 32D/neo () and 32D/Bcl-2 () cell suspensions were treated with vehicle or SC-9 and divided into two aliquots at the time of harvesting. A, quantitation of apoptotic nuclei. Expression of Cdc2 (B) and IGF-1 receptor (C). Lysates were immunoblotted with anti-Cdc2 and anti-IGF-1 receptor antibodies as described in Materials and Methods. Protein bands were quantitated by densitometric scanning of the X-ray films, normalized to band intensity in untreated control samples, and expressed as percentage of control for each time point. Data are the averages of at least three independent experiments. Vertical bars represent S.E.M.

Effects of SC-9 on IGF-1 Receptor Autophosphorylation. To test the hypothesis that SC-9 affected IGF-1 signaling at the receptor level, we attempted to detect IGF-1 receptor tyrosine phosphorylation as previously described (Vogt et al., 1998). Unfortunately, due to low phosphotyrosine levels, Western blot analysis of either lysates or IGF-1 receptor immunoprecipitates from 32D cells failed to indicate the presence of tyrosine-phosphorylated IGF-1 receptors in exponentially growing cells. Thus, we used LISN C4 cells, an NIH 3T3 cell line engineered to ectopically express high levels of the human IGF-1 receptor (Altschuler et al., 1994). IGF-1 stimulation of serum-starved cells resulted in increased IGF-1 receptor tyrosine phosphorylation (data not shown). Inclusion of a pharmacologically relevant concentration of SC-9 at 4 h before IGF-1 stimulation did not inhibit tyrosine phosphorylation in response to IGF-1, indicating that SC-9 did not directly interact with the IGF-1 receptor or the growth factor itself (data not shown). We have, however, seen inhibition of IGF-1 receptor autophosphorylation at higher concentrations of SC-9 (100 µM) or after a 6-h exposure (data not shown).

SC-9 Has In Vivo Antitumor Activity. Because SC-9 has apoptotic activities that are independent of IGF-1, a property not shared with other apoptosis-inducing agents, we examined the antitumor effects of SC-9 in a whole animal model. We used the in vivo excision clonogenic SCCVII tumor cell survival assay because of the relatively small amount of drug required for antitumor studies and because it permits a quantitative estimation of in vivo cell kill (Johnson et al., 1993). We first determined that the murine SCCVII squamous cell carcinoma tumor cells grown in culture were sensitive to SC-9. Figure 8A shows that SC-9 inhibited SCCVII cell proliferation with an IC₅₀ value of 20 µM, which is similar to the IC₅₀ value observed in mouse embryonic fibroblasts transformed with simian virus 40 large T antigen but lower than that seen with human breast cancer cells (Vogt et al., 1998). We then examined the antitumor activity of SC-9 in mice bearing established SCCVII tumors. A single i.p injection of SC-9 decreased tumor cell viability in a dose-dependent manner (Fig. 8B). The IC₅₀ value for SC-9 was 35 mg/kg; the maximum cell kill was a 60% decrease at 45 mg/kg (Fig. 8B). In comparison, a single dose of ~2.5 mg/kg cis-diamminedichloroplatinum (cisplatin) or 25 mg/kg carboplatin was required for a 50% reduction in SCCVII tumor clonogenicity. Thus, SC-9 had significant antitumor activity and was ~70% as potent as the clinically used carboplatin.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Growth inhibition and antitumor activity of SC-9 against SCCVII mouse small cell squamous carcinoma. A, growth inhibition assay. SCCVII cells were plated at 2000 cells/well in 96-well plates and treated with SC-9 for 48 h. Cell survival was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay as described in Materials and Methods. B, tumor excision clonogenic assay. Mice bearing SCCVII murine small cell carcinomas were treated with a single dose of SC-9 (), cisplatin (), or carboplatin (). After 24 h, tumors were excised, and a single cell suspension was prepared. Cell survival was scored with a clonogenic assay and expressed in surviving fraction per gram of tumor. There were three or four mice in each group with points representing means ± S.D.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

SC-9 is a simple, nonelectrophilic, small-molecule antiproliferative agent that was identified in a targeted array library modeled after complex natural product phosphatase inhibitors. This novel agent has antiproliferative activity against cancer cell lines in culture and is selective for the transformed phenotype. SC-9 is a potent inhibitor of Cdc25 and PTP1B in vitro (Rice et al., 1997). SC-9 also decreased Cdc2 protein levels (Vogt et al., 1998), a downstream event in the IGF-1 receptor-signaling cascade (Surmacz et al., 1992). The antiproliferative effects of SC-9, however, have not yet been directly linked to any of these actions. In this report, we demonstrate that SC-9, which is structurally unrelated to any known anticancer agent, caused Bcl-2 sensitive, but IGF-1 insensitive apoptosis in a well established model cell system and effectively sacrificed murine tumor cells in vivo. Furthermore, we have extended our investigations of the IGF-1-related effects of SC-9 to prove a functional involvement of IGF-1-mediated survival in SC-9-induced apoptosis.

Numerous recent reports have established a role for IGF-1 and its receptor in the establishment of the transformed phenotype (for review, see Resnicoff and Baserga, 1998). Auto/paracrine stimulation by IGF-1 is thought to play a major role in transformation by viral oncogenes (Baserga, 1993). High levels of circulating IGF-1 have been associated with an increased risk of prostate cancer (Chan et al., 1998). Studies in cultured cells have demonstrated that IGF-1 was able to protect cells from a variety of apoptotic stimuli, including the clinically used antineoplastic agents camptothecin, 5-fluorouracil, tamoxifen, and methotrexate (Dunn et al., 1997), as well as cisplatin and doxorubicin (Geier et al., 1995), suggesting that IGF-1 also may play a role in resistance to cancer chemotherapy.

Several approaches aimed at IGF-1 receptor inactivation have been shown to reverse the transformed phenotype, among them treatment with antisense oligonucleotides (Resnicoff et al., 1995b), deletion of the IGF-1 receptor gene by homologous recombination (Sell et al., 1994), or peptide analogs that interfere with autocrine IGF-1 receptor stimulation (Pietrzkowski et al., 1993; Hayry et al., 1995). Despite the large body of evidence that IGF-1 and the IGF-1 receptor are rational targets for anticancer drug design, few small-molecule-, cell-permeable-, specific disrupters of IGF-1 signaling have been identified to date. There are, however, examples of agents that specifically inhibit PI(3)K, a downstream event in the IGF-1 signaling pathway, some of which have shown promising antitumor activity (Norman et al., 1996).

In contrast to other growth factors, IGF-1 is also a potent antiapoptotic factor. To investigate a functional role of IGF-1 or its receptor in SC-9 toxicity, we chose 32D mouse myeloid progenitor cells as a well established model for apoptotic death, and found that SC-9 caused concentration-dependent apoptosis in these cells. Consistent with previous reports (Rodriguez-Tarduchy et al., 1992), IGF-1 protected 32D cells against apoptosis induced by cytokine withdrawal, but did not rescue cells from SC-9-induced apoptosis. IGF-1 also afforded protection against the apoptotic effects of etoposide, an agent whose mechanism of action is not known to involve inhibition of growth factor signaling. Furthermore, IGF-1 maintained or elevated expression levels of Cdc2 in the absence of IL-3 and during etoposide treatment, but not after treatment with equitoxic concentrations of SC-9. It should be noted, however, that IGF-1 protection from apoptosis was not complete and was only observed at high concentrations of IGF-1, perhaps because IGF-1 delays apoptosis rather than prevents it. Although all IGF-1 protection experiments were scored 24 h after exposure to apototic stimuli, it is possible that the antiapoptotic effects of IGF-1 could be more pronounced at earlier time points. We then examined whether overexpression of the IGF-1 receptor rendered cells less sensitive to SC-9 and found that SC-9-induced apoptosis was independent of IGF-1 receptor levels. The cells were, however, protected from apoptosis by SC-9 by overexpression of the antiapoptotic gene product Bcl-2. Bcl-2 has been shown to protect against a number of apoptotic stimuli such as chemotherapeutic drugs, irradiation, or growth factor deprivation but it does not prevent cell death by complement-mediated lysis, tumor necrosis factor-, or hydrogen peroxide, which often induce necrotic rather than apoptotic cell death (Reed, 1994). Bcl-2 prolongs cell survival without inducing proliferation, presumably by blocking a final common pathway in the execution of programmed cell death. Bcl-2 protection against SC-9 suggests that the compound acts on a specific intracellular target which, in turn, triggers the apoptotic machinery. Furthermore, although ectopic expression of Bcl-2 prevented apoptosis, it did not prevent down-regulation of Cdc2 in the presence of SC-9, indicating that the decrease in Cdc2 levels was a direct result of compound treatment and not secondary to cell death. Because Cdc2 is a biochemical indicator of IGF-1 signaling, these data suggest that SC-9 perturbs the IGF-1 receptor pathway. Whether the decrease in Cdc2 levels is important for programmed cell death by SC-9 is currently not known. Future experiments should focus on this issue.

The most obvious explanation for the above-mentioned results would be that SC-9 acts at the level of the IGF-1 receptor itself. Receptor autophosphorylation studies in NIH 3T3 cells, however, indicated that a short-term exposure to SC-9 did not affect IGF-1 tyrosine phosphorylation, but that concentrations higher than those inducing apoptosis or prolonged exposure times were required for a decrease in receptor autophosphorylation. Several important conclusions can be drawn from these data. First, SC-9 does not bind to or inactivate the growth factor itself, as is the case with suramin (Middaugh et al., 1992). Second, SC-9 does not bind to the extracellular portion of the IGF-1 receptor. Third, it is unlikely that SC-9 directly inhibits receptor tyrosine kinase activity. This is consistent with its lack of in vitro inhibition of Cdc2 (Rice et al., 1997), or c-Src tyrosine kinase (data not shown). Thus, it seems most likely that the molecular target of SC-9 is intracellular and distal to the IGF-1 receptor.

In summary, SC-9 is a novel inducer of apoptosis whose effects, in contrast to etoposide or IL-3 withdrawal, could not be overcome by high levels of exogenous IGF-1 or by overexpression of the IGF-1 receptor. Unlike IL-3 withdrawal, SC-9 also compromised the ability of IGF-1 to maintain levels of Cdc2, a downstream effector of an intact IGF-1 receptor pathway. These data suggest a functionally significant involvement of the IGF-1 receptor pathway in SC-9-induced apoptosis. The ability of SC-9 to counteract IGF-1 action in whole cells is apparently not due to inhibition of the IGF-1 receptor tyrosine kinase, but more work is needed to more precisely determine the molecular target(s) of SC-9. Even though SC-9's mechanism of action is not completely understood, its independence from growth factor-mediated survival sets SC-9 apart from other structurally and mechanistically unrelated antineoplastic agents such as antimetabolites (methotrexate), anthracyclines (doxorubicin), antiestrogens (tamoxifen), or topoisomerase inhibitors (camptothecin, etoposide), all of which are sensitive to the survival effects of IGF-1 or the IGF-1 receptor. Coupled with SC-9's selective in vitro activity for transformed cells and promising antitumor activity in an in vivo tumor model that is positive for the IGF-1 receptor, our data suggest that SC-9 or related compounds warrant further study as potential agents for the treatment of growth factor-dependent tumors.