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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Laboratory of Oncologic Research, Orthopaedic Rizzoli Institute; 2 Cancer Research Section, Department of Experimental Pathology, University of Bologna, Bologna, Italy and 3 Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland
Requests for reprints: Katia Scotlandi, Laboratory of Oncologic Research, Orthopaedic Rizzoli Institute, Via Di Barbiano 1/10, 40136 Bologna, Italy. Phone: 39-51-6366760; Fax: 39-51-6366761; E-mail: katia.scotlandi{at}ior.it.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we therefore chose to investigate the effects of a novel small molecule IGF-IR kinase inhibitor, NVP-AEW541 (35), a pyrrolo[2,3-d]pyrimidine derivative highly selective against IGF-IR, compared with the insulin receptor and other tyrosine kinases, on the growth of musculoskeletal tumors, including Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma. In vitro effects of the compound in association with conventional drugs currently used in the treatment of these tumors were also analyzed to identify best drug combinations.
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Materials and Methods |
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Drugs. NVP-AEW541 was kindly provided by Novartis Pharma (Basel, Switzerland). Stock solution of this drug was prepared in DMSO and stored at –20°C. Doxorubicin, cisplatin, methotrexate, vincristine, and actinomycin D were purchased from Sigma. D-18851, an ifosfamide analogue not requiring metabolic activation (22), was kindly provided by Baxter Oncology GmbH (Frankfurt, Germany). Working dilutions of all drugs were prepared immediately before use.
In vitro cytotoxicity. To study the effects of NVP-AEW541 in standard or low-serum conditions, 20,000 to 100,000 cells were plated into 24-well plates in IMDM plus 10% FBS. After 24 hours, medium was replaced by IMDM plus 10% FBS or 1% FBS with or without (control) various concentrations of the compound (30 nmol/L-3 µmol/L, Novartis Pharma) up to 6 days. Effects of the neutralizing antibody anti-IGF-IR 
IR3 (1 µg/mL, Calbiochem, San Diego, CA) were also evaluated in the same conditions as a comparison. Cell growth inhibition by daily administration of NVP-AEW541 was also considered. IC50 (drug concentration resulting in 50% inhibition of growth) values were determined by seeding 20,000 cells/cm2 in standard medium (IMDM + 10% FBS). After 24 hours, increasing doses of the drug were added. To evaluate the ability of IGF-I to induce cell recovery from the cytotoxic effects of NVP-AEW541, TC-71 cells were exposed to 300 nmol/L and 1 µmol/L NVP-AEW541 (corresponding to IC50 value) and 50 ng/mL IGF-I (Upstate Biotechnology Inc., Lake Placid, NY) for 48 hours. In all experiments, cell growth was evaluated on harvested cells by trypan blue vital cell count to estimate the percentage of growth inhibition compared with cells treated with DMSO-containing medium. Final concentration of DMSO in the medium was <0.001%, and in the present study, it had no effect on cell growth inhibition.
Combined in vitro treatments with NVP-AEW541 and conventional chemotherapeutics. 20,000 cells/cm2 of TC-71 Ewing's sarcoma cell line were seeded in IMDM plus 10% FBS. After 24 hours, cells were treated with varying concentrations of doxorubicin (range, 0.3-10 ng/mL), cisplatin (range, 3-300 ng/mL), vincristine (0.01-1 ng/mL), actinomycin D (range, 0.01-1 ng/mL) and ifosfamide analogue D-18851 (range, 10 ng/mL-1 µg/mL) without (control) or with NVP-AEW541 (100 nmol/L, corresponding to the dose that gives 
20% to 25% growth inhibition in TC-71 cell line). After 72 hours of treatment, cell growth was evaluated as previously described.
Cell cycle analysis. After 24 to 72 hours of treatment, cell cultures were incubated with 10 µmol/L bromodeoxyuridine (Sigma) for 1 hour in CO2 atmosphere at 37°C. Harvested cells were fixed in 70% ethanol for 30 minutes. After DNA denaturation with 2 N HCl, 1 x 106 cells were processed for indirect immunofluorescence staining using -bromodeoxyuridine monoclonal antibody diluted 1:4 as a primary antibody (Becton Dickinson, San Jose, CA) and analyzed by flow cytometry (FACSCalibur, Becton Dickinson). For analysis of DNA content, cells were fixed with cold 70% ethanol, treated with 0.5 mg/mL RNase, and stained with 20 µg/mL propidium iodide.
Analysis of apoptosis. For morphologic assessment of apoptotic nuclei, sarcoma cells were seeded in 60 mm dishes in IMDM plus 10% FBS. The following day, medium was changed in IMDM plus 10% FBS without (control) or with NVP-AEW541 (100 nmol/L-3 µmol/L). Twenty-four to 72 hours from treatment, cells were fixed in methanol/acetic acid (3:1) for 15 minutes and stained with 50 ng/mL Hoechst 33258 (Sigma). Detection and quantification of apoptotic cells was also obtained by flow cytometric analysis of Annexin V-FITC-labeled cells. This test was done according to the manufacturer's instructions.
Soft agar assay. Anchorage-independent growth was determined in 0.33% agarose (SeaPlaque, FMC BioProducts, Rockland, ME) with a 0.5% agarose underlay. Cell suspensions (cells per 60 mm ø dish: 3,300-10,000 for TC-71, SK-N-MC, and U-2OS; 33,000-100,000 for the other cell lines) were plated in a semisolid medium (IMDM + 10% or 1% FBS containing 0.33% agarose) with or without NVP-AEW541 (100 nmol/L-3 µmol/L). Dishes were incubated at 37°C in a humidified atmosphere containing 5% CO2, and colonies were counted after 7 to 15 days. Colonies with >50 cells were considered. Percentage of growth inhibition was calculated with respect to cells treated with vehicle alone.
Western blotting. Constitutive activation of IGF-IR was evaluated on a panel of Ewing's sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines. To analyze the effects of NVP-AEW541 compound on IGF-IR signaling pathway of TC-71, starved cells were pretreated for 2 hours with 300 nmol/L to 1 µmol/L NVP-AEW541 and then exposed to IGF-I (50 ng/mL, 5-60 minutes). In a second experiment, we followed NVP-AEW541 inhibitory effects on IGF-IR-related signaling pathways by exposing TC-71 to 300 nmol/L and 1 µmol/L of compound for 1 to 48 hours in standard medium. To determine phosphorylation status of Erk and Akt, two downstream mediators of mitogen-activated protein kinase (MAPK) kinase/MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, cell lysates were prepared with a buffer containing 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 0.1% SDS, 1% Triton X-100, 5 mmol/L EDTA, 1% deoxycholate, and protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate). Protein concentration was determined by Bio-Rad protein assay (Hercules, CA) and equivalent amounts of total cell lysate (50 µg) were separated by 7.5% or 10% SDS-PAGE under denaturating conditions and transferred onto nitrocellulose membrane. Membranes were incubated overnight with primary antibodies [anti-phospho-IGF-IR (Tyr1131) dilution 1:200, anti-phospho-Akt (Ser473) dilution 1:1,000, anti-phospho-p44/p42 MAPK (Thr202/Tyr204) dilution 1:1,000, anti-IGF-IR dilution 1:1,000, anti-Erk dilution 1:1,000, and anti-Akt dilution 1:1,000 (New England Biolabs, Cell Signaling Technology, Beverly, MA)] and then incubated with secondary anti-rabbit antibody conjugated to horseradish peroxidase (Amersham, Buckinghamshire, United Kingdom; dilution 1: 1,500). Membranes were revealed by enhanced chemiluminescence Western blotting detection reagents (Amersham). For analysis of IGF-IR tyrosine phosphorylation status by immunoprecipitation, total cell lysate (500 µg) was incubated with 1.5 µg specific anti-IGF-IR ß-subunit monoclonal antibody (clone C-20, Santa Cruz, San Diego, CA) overnight. Protein G-Sepharose (40 µL, Calbiochem) were then added and incubation continued for 2 hours. Protein G-Sepharose was collected, washed thrice with lysis buffer, and resuspended in SDS-gel sample buffer. Western blotting was done using anti-phospho-tyrosine antibody (clone Py20, BD Biosciences, San Diego, CA) followed by a secondary horseradish peroxidase–linked anti-mouse Ig antibody. Membranes were reprobed with anti-IGF-IR ß-subunit antibody.
In vivo treatments with NVP-AEW541 alone or in combination with vincristine. Female athymic 4- to 5-week-old Crl:C-1-nu/nu BR mice (Charles River Italia, Lecco, Italy) were used. Mice were treated according to institutional and European Union guidelines. Tumor growth was determined after s.c. injection of 5 x 106 TC-71 cells. Mice were randomized into controls and three treated groups when tumors started to be measurable (7 days after cell inoculation, day 0 of treatment). In the group treated with NVP-AEW541 alone, each mouse received 50 mg/kg dissolved in 25 mmol/L L(+)-tartaric acid p.o. twice daily, 7 days a week. The second group received vincristine i.p. (1 mg/kg/d) on days 0 and 1 of treatment. The third group received either NVP-AEW541 p.o. or two i.p. injections of vincristine following the time schedule mentioned above. The control group was treated p.o. with 25 mmol/L L(+)-tartaric acid only.
Tumor growth was assessed thrice weekly by measuring tumor volume, calculated as 
/2 · [
(ab)]3 / 6, where a is maximal tumor diameter and b is tumor diameter perpendicular to a. For ethical reasons, mice with tumor were sacrificed when they reached a tumor volume of 2.5 mL. Otherwise, mice were sacrificed 5 months after cell inoculation by CO2 inhalation and necropsied.
Statistical analysis. Differences among means were analyzed using a two-sided Student's t test. IC50 for each particular drug was defined as concentration of drug that reduces growth by 50% compared with untreated control cells and was calculated from linear transformation of dose-response curves. Analysis of drug combination effects was done by using the fractional product method.
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Results |
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A time course evaluation of inhibitory effects of 300 nmol/L NVP-AEW541 on MAPK and PI3K signaling pathways in standard medium, however, revealed transient effects on MAPK pathway, particularly for the dose of 300 nmol/L, whereas PI3K pathway appeared to be blocked up to 48 hours (Fig. 2A). Consequently, we determined whether a daily in vitro administration of NVP-AEW541 gave a benefit in terms of growth inhibition. Figure 2B shows that similar inhibitory effects were obtained in TC-71 cells with single or a repeated treatment using NVP-AEW541. This indicates that the stable inhibition of PI3K pathway is sufficient to guarantee remarkable growth inhibitory effects of NVP-AEW541. Growth inhibitory activity of the compound was maintained for at least 72 hours after its removal (33% of growth inhibition with the dose of 300 nmol/L and 50.3% of growth inhibition with the dose of 1 µmol/L; P < 0.05).
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IR3 (1 µg/mL) and NVP-AEW541 (300 nmol/L) indicate a similar activity for the two agents in all cell lines here considered, further confirming the specificity of action of the kinase inhibitor (data not shown). Figure 3A shows IC50 values obtained for these cells. Ewing's sarcoma cells were more sensitive to NVP-AEW541, showing IC50 values at submicromolar doses. Rhabdomyosarcoma cell lines rank second with respect to drug sensitivity, whereas osteosarcoma cells confirm that they are more refractory to strategies targeting IGF-IR (11). Interestingly, among rhabdomyosarcoma, the three cell lines of alveolar origin (RH4, RH30, and RC2) show a level of sensitivity comparable with that of Ewing's sarcoma cells, whereas cell lines of embryonal origin are definitely less sensitive. The level of sensitivity does not correlate with the level of expression of IGF-IR, because all cell lines here considered express the receptor at similar level. However, when activation status of IGF-IR was examined, we found a correlation between constitutive phosphorylation of IGF-IR and level of sensitivity to NVP-AEW541 (Fig. 3B).
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20% growth inhibition after 72 hours. Combined treatment with NVP-AEW541 and vincristine, actinomycin D, or the ifosfamide analogue D-18851 resulted in a significantly enhanced inhibition of cell growth with respect to the therapeutic efficacy of these drugs alone (Fig. 6A). In contrast, when cells were exposed to NVP-AEW541 and doxorubicin or cisplatin concomitantly, a subadditive cytotoxic effect was generally observed. Fractional product method confirmed antagonism when cells were treated with NVP-AEW541 and doxorubicin or cisplatin concomitantly and additive effects when NVP-AEW541 were combined with vincristine, actinomycin D, and ifosfamide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-IR signaling is an attractive target for new therapeutic strategies in sarcoma based on its role in the pathogenesis and progression of these tumors (4–7, 13–18). Indeed, IGFs promote tumor growth, survival, and migration of these cells and, by inducing vascular endothelial growth factor-A production, may favor their blood supply essential for the progressive growth of primary malignancies and for the development of metastases (19–26). Impairment of IGF-IR functions was therefore found to substantially contribute to control of sarcoma malignancy, especially of Ewing's sarcoma. Indeed, very promising and convincing preclinical results were obtained using a variety of approaches targeting IGF-IR, which include neutralizing antibodies, antisense IGF-IR RNAs, and competitive inhibitors, such as dominant-negative mutants. However, none of these approaches may be promptly applied in clinical research. In this respect, the recent availability of a selective small molecule, inhibiting IGF-IR signaling, raises new prospective for clinical studies. The compound, a pyrrolo[2,3-d]pyrimidine derivative, was recently presented as an optimized IGF-IR kinase inhibitor that selectively distinguishes at the cellular level between the native IGF-IR and the closely related insulin receptor. Its effects on other protein tyrosine kinases have been excluded, confirming the specificity of action (35). NVP-AEW541 shows a 27-fold inhibitory selectivity for the IGF-IR versus the insulin receptor (35) and is slightly more active (IC50 = 0.086 mol/L, toward IGF-IR kinase) and more selective than another pyrrolo[2,3-d]pyrimidine derivative that was recently studied in a series of tumor cell lines (39, 40). In addition, NVP-AEW541 has the advantage of being an orally bioavailable tyrosine kinase small molecule inhibitor. Considering the high degree of sequence identity between the IGF-IR and the insulin receptor, the high selectivity toward the IGF-IR is a major point of attractiveness and fulfills the need for agents that exhibit selectivity for IGF-IR versus the insulin receptor (2). Therefore, the compound has all the prerequisites for being considered a potential new drug for sarcomas, provided its effectiveness is proven and best treatment modalities are identified.
NVP-AEW541 was found to fulfill the key features expected from an IGF-IR inhibitor. It selectively inhibits IGF-I-mediated growth and signal transduction in Ewing's sarcoma cells. Of note, the inhibitory effects of NVP-AEW541 were not reverted in the presence of exogenous IGF-I. We believe that this is a major point because sarcoma cells, which always express IGF-IR, are likely to be locally exposed to paracrine and autocrine stimulation by IGF-I. In fact, IGF-I is normally stored in high quantity in bone matrix (41) and may be easily released by osteolysis induced by growing sarcoma cells. NVP-AEW541 shows higher growth inhibitory effectiveness in soft agar than in monolayer conditions, in keeping with the notion that IGF-IR is the key for growth of tumor cells under anchorage-independent conditions, but less for their growth as adherent monolayer. Comparing the effectiveness of NVP-AEW541 among the three major pediatric sarcomas (i.e., Ewing's sarcoma, rhabdomyosarcoma, and osteosarcoma), we found a generally higher activity toward Ewing's sarcoma versus rhabdomyosarcoma and, above all, osteosarcoma. Analysis of the constitutive activation status of IGF-IR in sarcoma cells indicated a correlation between level of phosphorylation of the receptor and sensitivity to the compound, indicating that the evaluation of phosphorylated level of IGF-IR is a valuable predictor of response to NVP-AEW541. In addition, Ewing's sarcoma cells are more dependent on IGF-IR functions for growth, survival, and migration (5) than rhabdomyosarcoma and osteosarcoma, which show a redundancy of autocrine loops (11, 12). Analysis of the effects of NVP-AEW541 on cell cycle and apoptosis on a panel of cell lines with a spectrum of sensitivity toward the compound revealed that NVP-AEW541 determines blockage of cells in G1 phase in all cell lines, whereas apoptotic effects were observed only in those cells that show a high level of sensitivity. This different activity of NVP-AEW541 seems to be due to a differential effect of the compound on intracellular signaling pathways. In fact, in cells that are sensitive to NVP-AEW541, such as the TC-71 Ewing's sarcoma cell line, the compound efficiently inhibited Akt activation in standard medium (i.e., in the presence of serum), whereas in cell lines that are less sensitive NVP-AEW541 had minimal effect on Akt activity (data not shown). These data provide further evidence that cell lines that are particularly sensitive to NVP-AEW541 are highly dependent on IGF-I for activation of critical signaling pathways. In addition, these findings suggest that efficient inhibition of PI3K-Akt signaling is a prerequisite for growth inhibition. Indeed, when a time course analysis of the effects of NVP-AEW541 on MAPK and PI3K signaling was done, we observed a transient inhibitory effect on Erk phosphorylation but a stable inhibition of Akt, in keeping with the inhibitory effects on cell growth.
As a final step, we investigated in vitro growth effects of NVP-AEW541 in combination with conventional therapeutic agents that are currently used in the treatment of Ewing's sarcoma, osteosarcoma, or rhabdomyosarcoma. It is now generally accepted that IGF-I attenuates the response of cancer cells to several chemotherapeutic agents (1, 2). Thus, inhibition of IGF-I action could be a useful adjuvant to cytotoxic chemotherapy. Our in vitro findings illustrate that the compound may be advantageously used in combination with vincristine, actinomycin D, and ifosfamide but not with doxorubicin and cisplatin. The agonistic effect of NVP-AEW541 and vincristine was also confirmed in the in vivo study. Significant inhibition of Ewing's sarcoma tumor growth was indeed observed only with combined treatments.
However, this observed synergistic in vivo effects of the IGF-IR inhibitor and vincristine in this s.c. model may not reflect the situation when the tumor localizes to the bone. Therefore, this drug merits further in vivo evaluation for what concerns distance metastases in a appropriate model prior to human studies.
In conclusion, we show that the availability of the selective IGF-IR kinase inhibitor NVP-AEW541 may be a promising approach in the treatment of Ewing's sarcoma. However, for the broadest applicability and best efficacy in sarcomas, NVP-AEW541 may be combined with vincristine, actinomycin D, and ifosfamide, three major drugs in the treatment of these tumors. In addition, NVP-AEW541 by showing a significant increase of the inhibitory effects of these drugs may reduce their toxicity allowing a decrease in drug dosage.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 9/ 7/04. Revised 2/ 2/05. Accepted 2/15/05.
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) or after treatments repeated every 24 hours (
). Cells were harvested after 72 hours.
) p.o. twice daily, 7 days a week for 2 weeks; vincristine alone i.p. (1 mg/kg/d) on days 0 and 1 of treatment (
); NVP-AEW541 p.o. alone twice daily, 7 days a week for 2 weeks (
); or NVP-AEW541 p.o. twice daily, 7 days a week + vincristine i.p. (1 mg/kg/d) on days 0 and 1 of treatment (
). Treatments began when tumors started being measurable. *, P < 0.05, Student's t test.