Inhibition of Insulin-like Growth Factor I Receptor Increases the Antitumor Activity of Doxorubicin and Vincristine against Ewing’s Sarcoma Cells¹

ES,³ which includes conventional ES, Askin’s tumor, and PNET, ranks second in frequency among primary bone tumors. It is an extremely aggressive, poorly differentiated neoplasm of uncertain histogenesis, usually arising in children and young adults. Combination chemotherapy associated with local control with surgery or radiation therapy have become standard practice in the treatment of patients with ES (1, 2, 3). However, despite the use of multimodal treatments and of very aggressive chemotherapeutic regimens, the long-term disease free survival of ES patients is still disappointingly low, particularly in high-risk groups (2, 4, 5). The identification of valuable new therapeutic targets for the design of innovative, more effective strategies is, therefore, urgently needed. In recent years, by analyzing the role of growth factors in the pathogenesis of ES, we and others have reported on the autocrine production of IGF-I and the constant presence of its corresponding receptor, the IGF-IR, in ES cells (6, 7). IGF-IR, a membrane-bound heterotetramer receptor with intrinsic tyrosine kinase activity, is emerging as an important factor in the regulation of cell growth in different tumor types, both in vitro and in vivo. In particular, IGF-IR, activated by its ligands, appears to regulate cell growth in at least three different ways: it is mitogenic; it is mandatory for the establishment and the maintenance of the transformed phenotype; and it protects cells from apoptosis. Targeting IGF-IR by using different approaches, including antisense technologies (8, 9, 10), antibodies against IGF-IR (11, 12), and dominant negative mutants of IGF-IR (13, 14, 15), resulted in the reversal of the transformed phenotype in several types of tumor cells. In ES, the blockage of IGF-IR-mediated circuit by αIR3 MAb, which specifically neutralizes IGF-IR greatly inhibits the growth and the migration ability of ES cells in vitro (6), as well as their tumorigenic and metastatic ability in vivo (16). A significant inhibition of ES growth has also been successfully achieved in vivo by using suramin, a nonspecific growth factor antagonist that inhibits a number of autocrine circuits, including the IGF-IR-mediated loop (17). Therefore, impairment of IGF-IR appears to be a valuable therapeutic approach against ES. Moreover, Toretsky et al. recently reported that ES cells use an IGF-IR initiated signaling pathway through phosphoinositide 3-OH kinase and Akt for survival when treated with doxorubicin (18), indicating in IGF-IR a possible mechanism of ES resistance to chemotherapy. In this study, we investigated whether impairment of IGF-IR by αIR3 neutralizing MAb and/or suramin may be advantageously combined with conventional cytotoxic drugs for the design of more effective therapeutic regimens. Although suramin does not exert a specific action against IGF-IR, in ES/PNET its effects substantially overlap those obtained with the specific neutralizing antibody anti-IGF-IR. This is probably attributable to the peculiar and unique presence, in this neoplasm, of a autocrine IGF-IR-mediated loop (7). Suramin ability to specifically block IGF-IR function in ES/PNET was demonstrated previously (16).

The ES cell line TC-71 was a generous gift from T. J. Triche (Childrens Hospital, Los Angeles, CA). The Askin’s tumor cell line SK-N-MC was obtained from the American Type Culture Collection (Rockville, MD). The PNET cell line IOR/LAP35 was previously established and characterized at the Istituti Ortopedici Rizzoli. Cells were routinely cultured in IMDM (Life Technologies, Inc., Paisley, Scotland), supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% inactivated FCS (Biological Industries, Kibbutz Beth Haemek, Israel). Cells were maintained at 37°C in a humidified 5% CO₂ atmosphere.

Cells (200,000) of TC-71 and SK-N-MC or 500,000 cells of IOR/LAP-35 were seeded in 6-well plates in IMDM plus 10% FCS. After 24 h, cells were treated with varying concentrations of doxorubicin (range 10 pg/ml–100 ng/ml) or vincristine (range 10 pg/ml–1ng/ml) without (control) or with the blocking MAb αIR3 (1 μg/ml; Calbiochem-Novabiochem Co., San Diego, CA) or suramin (250 μg/ml; kindly provided by Bayer AG, Leverkusen, Germany; 100 μg/ml was used for the SK-N-MC cell line because these cells are particularly sensitive to suramin). As an additional control, the isotype-matched control MAb MOPC-21 (1 μg/ml; Sigma Chemical Co., St. Louis, MO) was also used. After 96 h of treatment, cell growth was evaluated on harvested cultures by trypan blue vital cell count. For sequential treatments, cells were distributed into 6-well plates, and treatment started the following day. Doxorubicin or vincristine were added to appropriate wells, at the concentrations indicated in the figure legends. After 12–24 h, depending on the doubling time of the cell line, the drugs were removed by washing the cells twice, followed by the addition of cell culture medium plus αIR3 MAb (1 μg/ml) or suramin (250 μg/ml; 100 μg/ml was used for the SK-N-MC cell line, because these cells are particularly sensitive to suramin). After 96 h of continuous presence of αIR3 MAb or suramin, cell growth was evaluated on harvested cells by trypan blue vital cell count.

Five thousand to 10,000 TC-71 cells/cm² were seeded in IMDM plus 10% FCS. After 24 h, cells were treated with varying concentrations of doxorubicin (range, 13–100 ng/ml) either without (control) or with αIR3 MAb (1 μg/ml), or suramin (250 μg/ml), respectively. After 36 h from seeding, cell cultures were incubated with 10 μm BrdUrd (Sigma Chemical Co.) for 1 h in a CO₂ atmosphere at 37°C. Harvested cells were fixed in 70% ethanol for 30 min. After DNA denaturation with 2 n HCl for 30 min at room temperature, cells were washed with 0.1 m Na₂B₄O₇ (pH 8.5). Cells (10⁶) were then processed for indirect immunofluorescence staining, using α-BrdUrd (Euro-Diagnostics, Milan, Italy) diluted 1:4 as a primary MAb, and analyzed by flow cytometry (FACSCalibur, Becton Dickinson, Milan, Italy). For the cell cycle analysis, 70% ethanol-fixed cells were pretreated with 100 μg/ml RNase for 30 min at 37°C and stained with 20 μg/ml propidium iodide before flow cytometric analysis.

TC-71 Cells were seeded and treated with doxorubicin with or without αIR3 MAb as reported for BrdUrd labeling index. After a 24–96 h of in vitro treatment, cells were fixed in methanol/acetic acid (3:1) for 15 min, and stained with 50 ng/ml hoechst 33258 (Sigma Chemical Co.). Cells with three or more chromatin fragments were considered as apoptotic. The percentage of apoptotic nuclei was evaluated based on 1000–2000 nuclei.

Anchorage-independent growth was determined in 0.33% agarose (SeaPlaque; FMC BioProducts, Rockland, ME) with a 0.5% agarose underlay. TC-71 cell suspensions (1000–3300 cells/dish) were plated in a semisolid medium (IMDM plus 10% FCS containing 0.33% agarose) with or without varying concentrations of doxorubicin (range 3–100 ng/ml) or vincristine (300 pg/ml–1 ng/ml) plus anti-IGF-IR αIR3 MAb (1 μg/ml). The MAb MOPC-21 (1 μg/ml) was also used as an additional control. Dishes were incubated at 37°C in a humidified atmosphere containing 5% CO₂, and colonies were counted after 7 days.

Differences among means were analyzed using a two-sided Student’s t test. The analysis of drug combination effects was performed by using the fractional product method.

Experiments were carried out to determine the effect on the growth of ES/PNET cells of two conventional chemotherapeutic drugs (doxorubicin or vincristine) in combination with anti-IGF-IR MAb αIR3 or suramin. Continuous treatments with either αIR3 MAb or suramin produced a dose-dependent inhibition of growth (7, 16). The combined treatment with increasing concentrations of doxorubicin or vincristine and 1 μg/ml of αIR3 MAb resulted in a synergistic or additive inhibition of TC-71 cell growth with respect to the therapeutic efficacy of doxorubicin or vincristine alone, respectively (Fig. 1, A and B). The increased antitumor activity of anticancer agents was not observed when cells were treated with chemotherapeutic drugs in combination with MOPC-21 MAb, an isotype-matched MAb, used as a control. An additive cytotoxic effect was also obtained when doxorubicin or vincristine was used in combination with suramin (Fig. 2,A and B). Table 1 summarizes the increased sensitivity to doxorubicin or vincristine of three cell lines representative of the whole spectrum of ES/PNET tumors, including conventional ES (TC-71), PNET (LAP-35), and Askin’s tumor (SK-N-MC), after combined simultaneous treatments. To closely mimic the in vivo treatment conditions, we performed sequential treatments. In particular, ES/PNET cells were exposed to doxorubicin or vincristine for only 12–24 h, a time that corresponds to the in vitro doubling time of these cell lines, and then were maintained in the presence of αIR3 MAb or suramin for an additional 96 h. Again, a synergistic or additive inhibitory effect on TC-71 cell growth was observed (Fig. 3, A and B; Fig. 4, A and B) and the sensitivity of ES/PNET cells to doxorubicin or vincristine was significantly enhanced with combined sequential treatments (Table 2). These effects occurred at clinically relevant drug concentrations. The in vitro conditions closely simulated drug plasma levels that may persist for a prolonged period and are sufficient to kill tumor cells without severe toxic effects (17, 19, 20).

To ascertain whether the inhibitory effect on TC-71 cell growth in vitro was attributable to IGF-IR impairment on the cell cycle and/or induction of apoptosis, the incorporation of BrdUrd and the percentage of cells in the different phases of cell cycle, as well as the percentage of apoptotic nuclei, were analyzed after treatment with different doses of doxorubicin alone or in association with αIR3 MAb (1 μg/ml) or suramin (250 μg/ml). Doxorubicin progressively induced a consistent reduction in the G₁ and S-phase rate, as well as an enhancement in the percentage of cells in G₂-M phase of the cell cycle. In combined treatments, αIR3 MAb and suramin significantly increased the G₁-phase rate, only partly affected the doxorubicin-induced decrease of BrdUrd-positive cells, and failed to significantly modify the G₂-M blockage induced by doxorubicin (Fig. 5). With regard to the percentage of apoptotic nuclei after treatment with αIR3 MAb or with suramin and doxorubicin, a synergistic effect was observed in the combined treatment using αIR3, whereas the association of suramin with doxorubicin gave an additive increase in the apoptotic rate observed with doxorubicin alone (Fig. 6, A and B). These findings confirmed the hypothesis that the specific blockage of IGF-IR deprives ES cells of an important tool for preventing apoptosis that is induced by chemotherapeutic agents.

Treatment of TC-71 cells with increasing concentrations of doxorubicin induced a progressive inhibition in the number of colonies observed in semisolid medium (Table 3). Combined treatments of doxorubicin plus αIR3 MAb synergistically increased growth inhibition induced by doxorubicin alone, whereas the combined treatment with vincristine had an additive effect. These findings appeared to be specific for the blockage of IGF-IR, because MOPC-21 MAb did not affect the cytotoxic effect of doxorubicin.

In recent years, treatment modalities based on growth factor-receptor interactions or on interference with their signal transduction pathways seemed to be promising therapeutic possibilities, especially for those neoplasms such as ES in which conventional chemotherapy, although successful in many cases, has clearly shown an impasse (2). In previous studies, we found that IGF-IR signal transduction plays a critical role in the regulation of in vitro and in vivo ES cell growth (7, 16). In particular, impairment of IGF-IR functions by using a specific neutralizing MAb or suramin, a drug that is able to disrupt IGF-I binding from its receptor (17), resulted in a loss of tumorigenic and metastatic ability of ES cells in nude mice (16). Targeting IGF-IR seems, therefore, to be of potential therapeutic help in the treatment of ES patients. However, from a clinical point of view, to be of significant therapeutic value, MAbs against IGF-IR and suramin should be effectively combined with anticancer drugs in mediating their antitumor activity. The present study showed that combined treatments using IGF-IR inhibiting agents (both αIR3 MAb or suramin) in association with conventional cytotoxic agents, such as doxorubicin and vincristine, currently included in all ES/PNET chemotherapeutic regimens, significantly enhance the in vitro cytotoxic effects of chemotherapeutic drugs.

Most anticancer agents, including doxorubicin and vincristine, kill cancer cells by inhibiting cell cycle and/or inducing apoptosis (21, 22, 23). The apoptotic action of chemotherapeutic drugs has been extensively studied in the last few years, and it has been claimed to be the main mechanism of action. On the other hand, IGF-IR, activated by its ligands, is emerging as a powerful inhibitor of apoptosis induced by a variety of agents, including anticancer drugs and ionizing and nonionizing radiation (24, 25, 26, 27). In particular, Dunn et al. (27) first showed that IGF-I could induce a 20–40% increase in cell survival of breast cancer cells treated with clinically relevant and functionally diverse anticancer drugs, which supported the idea that IGF-I may significantly contribute to decreasing the effectiveness of chemotherapy. In ES, a recent study clearly indicates that IGF-I can act as a survival factor and that the activation of IGF-IR makes cells more resistant to doxorubicin-induced apoptosis (18). Therefore, blockage of IGF-IR functions by using a neutralizing MAb or suramin might sensitize ES cells to apoptosis that is induced by chemotherapeutic drugs, potentiating in this way their cytotoxic action. Indeed, by targeting IGF-IR, we observed an additive or, in some cases, a synergistic effect of growth inhibition induced by doxorubicin or vincristine. The percentage of growth inhibition from simultaneous or sequential treatments with chemotherapeutic drugs plus αIR3 MAb or suramin was enhanced by 30–35% compared with the effects induced by anticancer agents alone. This percentage corresponds to the percentage of growth inhibition induced by αIR3 MAb or suramin as single agents. As may be expected, αIR3 MAb, which specifically blocks IGF-IR, generally gave better results than suramin. However, despite the specificity of αIR3 MAb, the potential of anticancer strategies based on IGF-IR blockage by delivery of a murine antibody are of limited practical value in clinical settings because of the emergence of immune responses, the short peptide half-life, and the high cost. The use of suramin may, therefore, be an attractive alternative.

The analysis of the cell cycle indicated that the blockage of IGF-IR induces a blockage of the cells in G₁ phase that joins the G₂-M blockage induced by doxorubicin. The mitogenicity of IGF-IR has been known for a long time (28), and, for an optimal growth, IGF-IR is required in all cell cycle phases. The inhibition of IGF-IR mitogenic activity diminishes cell entry into S phase, but this effect does not antagonize the action of anticancer agents, such as doxorubicin, on the cell cycle. Indeed, agents that block IGF-IR and doxorubicin appeared to act on different phases of the cell cycle, and targeting IGF-IR may potentiate the action of doxorubicin. On the other hand, the analysis of apoptotic nuclei showed that αIR3 MAb or suramin actually have a significant effect in enhancing the apoptotic effects of doxorubicin. These findings confirmed that the disruption of IGF-IR antiapoptotic action may potentiate in vitro drug-induced cell death. Because the proapoptotic effects attributable to targeting IGF-IR are more dramatic when cells are in anchorage-independent conditions (28), we ascertained the effects of a combination treatment in semisolid medium. αIR3 MAb treatment induced a highly significant increase in the anchorage-independent growth inhibition of TC-71 cells observed after doxorubicin exposure.

The ability of tumor cells to grow in the absence of contact with extracellular matrix should not be considered as an artifact of cultured cells: anchorage-independence correlates quite well with tumorigenicity (29) and is probably the property that allows tumor cells to infiltrate surrounding tissues and to establish distal metastases. In this context, the favorable effect derived from the inhibition of antiapoptotic signaling of IGF-IR, which renders the cells more sensitive to the apoptotic action of doxorubicin, might be particularly relevant.

Taken together, our findings indicate that therapeutic strategies aimed at the blockage of IGF-IR, by using either αIR3 MAb or suramin, could yield a potential advantage in combined treatments with conventional therapeutic agents for ES patients.

The authors thank Dr. Alba Balladelli (Laboratorio di Ricerca Oncologica, Istituti Ortopedici Rizzoli, Bologna, Italy) for critical reading of the manuscript.