Human Ewing Sarcoma Research Articles

Expression of granulocyte‐colony‐stimulating factor and its receptor in human Ewing sarcoma cells and patient tumor specimens

Ewing sarcoma (ES) is a highly vascular malignancy. It has been demonstrated that both angiogenesis and vasculogenesis contribute to the growth of ES tumors. Granulocyte-colony-stimulating factor (G-CSF), a cytokine known to stimulate bone marrow (BM) stem cell production and angiogenesis, is routinely administered to ES patients after chemotherapy. Whether ES cells and patient tumor samples express G-CSF and its receptor (G-CSFR) and whether treatment with this factor enhances tumor growth was examined. Human ES cell lines were analyzed for expression of G-CSF and G-CSFR in vitro and in vivo. Sixty-eight paraffin-embedded and 15 frozen tumor specimens from patients with ES were also evaluated for the presence of G-CSF and G-CSFR. The in vivo effect of G-CSF on angiogenesis and BM cell migration was determined. Using a TC/7-1 human ES mouse model, the effect of G-CSF administration on ES tumors was investigated. G-CSF and G-CSFR protein and RNA expression was identified in all ES cell lines and patient samples analyzed. In addition, G-CSF was found to stimulate angiogenesis and BM cell migration in vivo. Tumor growth was found to be significantly increased in mice treated with G-CSF. The average tumor volume for the group treated with G-CSF was 1218 mm(3) compared with 577 mm(3) for the control group (P = .006). The findings that ES cells and patient tumors expressed both G-CSF and its receptor in vitro and in vivo and that the administration of G-CSF promoted tumor growth in vivo suggest that the potential consequences of G-CSF administration should be investigated further.

360 POSTER Dasatinib (BMS-354825), a novel, potent inhibitor of Bcr-Abl and Src, has a significant migration effect on human neuroblastoma and Ewing sarcoma cells

360 POSTER Dasatinib (BMS-354825), a novel, potent inhibitor of Bcr-Abl and Src, has a significant migration effect on human neuroblastoma and Ewing sarcoma cells

Hypoxia responsive element regulated herpes simplex virus-thymidine kinase system enhances killing effect of gancyclovir on Ewing's sarcoma cell line under hypoxic condition

To find out a possible approach to improve the effectiveness of radiotherapy and chemotherapy for Ewing's sarcoma by constructing a eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia and to evaluate the effects of this HRE regulated HSV-TK system on killing effect of gancyclovir (GCV) on Ewing's sarcoma cell line SK-ES under hypoxic condition. The HRE was synthesized according to the literature and cloned into the enhancer site of pIRES(2)-EGFP vector to obtain the pHRE recombinant plasmid. The HSV-TK was amplified by PCR and cloned into the multiple clone site of pIRES(2)-EGFP and pHRE to obtain pTK and pHRE-TK recombinant plasmid. The human Ewing's sarcoma cell line SK-ES was transfected by pTK or pHRE-TK recombinant plasmid with liposome and then was exposed to normoxic (21% oxygen) or hypoxic (3% oxygen) condition. The expression of enhanced green fluorescent protein (EGFP) was monitored by fluorescent microscopy. The sensitivity of human Ewing's sarcoma cell line SK-ES transfected with pTK or pHRE-TK recombinant plasmid to the anti-tumour drug GCV was determined with the method of tetrazolium (MTT) after treating with GCV for five days. (1) The result of sequencing showed that the recombinant plasmid pHRE contained HRE, and that the recombinant plasmid pTK and pHRE-TK contained HSV-TK gene in the sense direction. (2) Comparison of fluorescent optical density (FOD) showed that (1) the EGFP FOD value of pHRE and pHRE-TK group cells exposed to hypoxia was significantly higher than those exposed to normoxia (P < 0.01); (2) when the cells were exposed to hypoxia, the EGFP FOD value of pHRE and pHRE-TK group cells was significantly higher than that of pTK and empty vector group (P < 0.01); (3) there was no significant difference among the four groups of cells when they were exposed to normoxia (P > 0.05). (3) Comparison of the sensitivity of four groups of cells to GCV showed that (1) the cells in pHRE-TK and pTK groups were much more sensitive to GCV than the cells in pHRE group under hypoxia condition (P < 0.01), the higher the GCV concentration, the greater the difference; (2) the cells of pHRE-TK group were more sensitive to GCV than those in pTK group under hypoxic condition (P < 0.01), but was almost equally sensitive under normoxic condition (P > 0.05); (3) the pHRE-TK group cells had higher sensitivity to GCV under hypoxia than normoxia (P < 0.01) while the pTK group cells had almost the same sensitivity to GCV under hypoxia and normoxia (P > 0.05). (1) The eukaryotic expression vector expressing herpes simplex virus-thymidine kinase (HSV-TK) regulated by hypoxia responsive element (HRE) under hypoxia was constructed successfully. (2) HRE could up-regulate expression of EGFP by SK-ES cells under hypoxia condition. (3) HRE could enhance the killing effect of HSV-TK/GCV system on human Ewing's sarcoma cell line SK-ES under hypoxic condition.

Characterization of receptors for growth hormone-releasing hormone in human osteosarcomas and Ewing's sarcomas

Antagonists of growth hormone-releasing hormone (GH-RH) inhibit growth of various human cancers including osteosarcomas and Ewing's sarcomas, xenografted into nude mice or cultured in vitro. The antiproliferative effect of GH-RH antagonists could be mediated, in part, through the splice variants (SVs) of receptors for GH-RH which have been found in several human cancers and cancer cell lines. In this study we investigated the expression of SVs of GH-RH receptors and the binding characteristics of these receptor isoforms in MNNG/HOS human osteosarcoma and SK-ES-1 human Ewing's sarcoma grown in nude mice. RT-PCR revealed the presence of mRNA for SVs of GH-RH receptors in both human malignant bone cancer models. Using ligand competition assays with 125I-labeled GH-RH antagonist JV-1-42, we demonstrated in MNNG/HOS and SK-ES-1 tumors the presence of specific high affinity binding sites for GH-RH (Kd=5.83 nM and Kd=2.76 nM) with a maximal binding capacity (Bmax) of 552.1 fmol/mg protein and 371.9 fmol/mg protein, respectively. We also investigated the effect of GH-RH antagonist JV-1-38, administered s.c. at a dose of 20 microg twice daily for 4 weeks on the gene expression, affinity and concentration of receptors for GH-RH in MNNG/HOS human osteosarcomas xenografted into nude mice. Treatment with JV-1-38 did not affect the expression and binding characteristics of GH-RH receptors. High affinity binding of JV-1-38 to GH-RH receptors on MNNG/HOS tumors was characterized by an IC50 value of 1.04 nM. The presence of GH-RH receptors in human bone tumors provides a rationale for new approaches to the therapy of this malignancy based on GH-RH antagonists.

Establishment and Characterization of a Clonal Human Extraskeletal Ewing's Sarcoma Cell Line, EES1

Ewing's sarcoma, a small round cell sarcoma arising in soft tissue as well as the bone, is one of the most malignant tumors in children and young adults. Few established cell lines of extraskeletal Ewing's sarcoma (EES) have been reported, which made it difficult to examine the biological features of EES. Therefore, we have established a new clonal cell line of EES. We report its morphological characters, results of chromosomal and immunohistochemical analysis. A piece of tumor obtained from the 18-year-old female patient with EES was xenografted in a nude mouse. In vitro subcultured cells were then obtained from this xenograft. A clonal cell line was subsequently established by limiting dilution and designated EES1. EES1 cells had a doubling time of 24 hours. In the xenografted tumor, the cells expressed vimentin, CD99 (MIC2), neuron specific enolase (NSE) and cytokeratin. The original tumor cells also expressed vimentin, CD 99, and NSE, but was negative for cytokeratin. The morphological and immunohistochemical features of this cell line established, except for cytokeratin expression, were consistent with those of the primary tumor. Cytogenetic analysis of EES1 revealed chromosomal translocation of t(11; 12)(q24;ql2). The chimeric fusion of the Ewing's sarcoma gene in band 22q12 with the Friend leukemia virus integration-1 gene in band 11q24 was also demonstrated. Fluorescence in situ hybridization further confirmed the presence of translocation involving the Ewing's sarcoma gene in both the primary tumor and EES1 cells. In conclusion, we have established a human EES cell line EES1, which will provide a useful model for studying various aspects of human EES.

Interleukin-12 Up-Regulates Fas Expression in Human Osteosarcoma and Ewing's Sarcoma Cells by Enhancing Its Promoter Activity

Interleukin-12 (IL-12) has shown significant antitumor activity in several preclinical animal tumor models. Our previous studies showed that IL-12 inhibited tumor growth in human osteosarcoma and Ewing's sarcoma animal model. Decreased Fas expression in osteosarcoma increased the lung metastatic potential. In this study, we further examined the mechanism of IL-12 antitumor activity and showed that IL-12 significantly increased Fas expression in both human osteosarcoma cells LM7 and Ewing's sarcoma cells TC71. Up-regulation of Fas expression increased their sensitivity to Fas-induced cell apoptosis. Constructs of the Fas promoter linked to a luciferase reporter gene were used to determine the promoter activity. IL-12 increased Fas promoter activity 4.2- and 4.9-fold in TC71 and LM7 cells, respectively. Time course studies have shown that recombinant IL-12 stimulated Fas promoter activity at 2 hours, reached the peak level at 4 hours, and then declined at 24 hours. To investigate whether IL-12 specifically enhanced Fas promoter activity, we determined whether another gene (E1A) was able to stimulate Fas promoter activity. We also evaluated effect of IL-12 on the topoisomerase IIalpha promoter. The results indicated that E1A but not IL-12 stimulated topoisomerase IIalpha promoter activity. E1A failed to increase Fas promoter activity. We also found that kappaB-Sp1 element at position -295 to -286 in Fas promoter was essential for IL-12-induced activation, and nuclear factor-kappaB transcription factor was activated after IL-12 treatment in TC71 cells. These results indicate that IL-12 up-regulates Fas expression in human osteosarcoma and Ewing's sarcoma by enhancing Fas promoter activity. Understanding this mechanism may lead to new therapeutic approaches for the treatment of sarcoma involving the use of IL-12.

Zoledronic acid inhibits primary bone tumor growth in Ewing sarcoma

Zoledronic acid has been shown to be effective in the treatment of osteoporosis, hypercalcemia, and metastatic bone tumors. The efficacy of zoledronic acid on primary bone tumors has not been investigated. A primary bone tumor mouse model was established. Intratibia injection of TC71 cells resulted in an osteolytic bone tumor. Four days after injection the mice were treated with zoledronic acid alone, paclitaxel alone, or zoledronic acid plus paclitaxel. Control mice were treated with phosphate-buffered saline. Bone tumor growth was assessed using a Faxitron Specimen Radiography System. The gene expression was detected by reverse-transcription polymerase chain reaction (RT-PCR), ELISA, and immunohistochemistry. Osteoclast formation was determined by tartrate-resistant acid phosphatase (TRAP) staining. Zoledronic acid induced apoptosis in TC71 human Ewing sarcoma cells and inhibited cell proliferation. Five weeks after injection, 89% of mice in the control group developed osteolytic bone tumors. Paclitaxel had little effect on bone tumor growth, with 78% of mice developing tumors. By contrast, 44% of mice treated with zoledronic acid developed bone tumors. The most effective treatment was zoledronic acid plus paclitaxel. Tumor incidence in the combination therapy group was only 22%. Osteoclasts were quantified using TRAP staining. There was a decrease in TRAP-positive osteoclasts in tumor tissues from zoledronic acid-treated animals compared to control animals. RT-PCR, immunohistochemistry, and ELISA assay demonstrated that zoledronic acid up-regulated osteoprotegerin expression. These results suggest that zoledronic acid induces apoptosis and inhibits primary bone tumor growth in Ewing sarcoma through a mechanism involving the up-regulation of osteoprotegerin. Zoledronic acid may provide a novel therapeutic approach for the treatment of patients with Ewing sarcoma.

Doxorubicin modulates telomerase activity in Ewing's sarcoma in vitro and in vivo

Since most tumours escape replicative senescence by re-activation of the enzyme telomerase, telomerase is a promising target in the treatment of cancer and a promising marker for diagnosis and therapeutic response. We evaluated the effects of doxorubicin, one of the most active drugs in the treatment of Ewing's sarcoma, on telomerase in the human Ewing's sarcoma cell line STA-ET-1 in vitro and in STA-ET-1 xenografts in vivo. Telomerase activity (TA) was examined by TRAP-assay and real-time PCR. Real-time PCR was also used to quantify the mRNA expression of the catalytic subunit of telomerase (hTERT). In vitro growth inhibition was determined by the MTT-assay. Tumour xenografts were analyzed for tumour volume, apoptosis, necrosis, and proliferation. Doxorubicin concentrations that inhibited in vitro growth of STA-ET-1 by 50% compared to untreated controls ranged between 0.14 microM after 24 h and 0.01 microM after 72 h. Compared to untreated controls doxorubicin reduced TA in STA-ET-1 at toxic concentrations, but increased TA at non-toxic concentrations. In comparison with untreated xenografts, TA was reduced to 65% and hTERT expression dropped to 25% within 72 h in xenografts treated with 17.5 mg/kg doxorubicin i.p.; both recovered to initial values after 264 h. The rate of proliferating cells dropped to 70% within 96 h and increased thereafter. The highest rates of necrosis and apoptosis were seen after 96 h. hTERT expression co-varied significantly with proliferation but not with TA, apoptosis, and necrosis. No correlation was observed between TA, proliferation, apoptosis and necrosis. The results suggest doxorubicin induces down-regulation of hTERT gene expression that at least in part modulates TA in these tumours.

The Oncogenic TLS-ERG Fusion Protein Exerts Different Effects in Hematopoietic Cells and Fibroblasts

The oncogenic TLS-ERG fusion protein is found in human myeloid leukemia and Ewing's sarcoma as a result of specific chromosomal translocation. To unveil the potential mechanism(s) underlying cellular transformation, we have investigated the effects of TLS-ERG on both gene transcription and RNA splicing. Here we show that the TLS protein forms complexes with RNA polymerase II (Pol II) and the serine-arginine family of splicing factors in vivo. Deletion analysis of TLS-ERG in both mouse L-G myeloid progenitor cells and NIH 3T3 fibroblasts revealed that the RNA Pol II-interacting domain of TLS-ERG resides within the first 173 amino acids. While TLS-ERG repressed expression of the luciferase reporter gene driven by glycoprotein IX promoter in L-G cells but not in NIH 3T3 cells, the fusion protein was able to affect splicing of the E1A reporter in NIH 3T3 cells but not in L-G cells. To identify potential target genes of TLS-ERG, the fusion protein and its mutants were stably expressed in both L-G and NIH 3T3 cells through retroviral transduction. Microarray analysis of RNA samples from these cells showed that TLS-ERG activates two different sets of genes sharing little similarity in the two cell lines. Taken together, these results suggest that the oncogenic TLS-ERG fusion protein transforms hematopoietic cells and fibroblasts via different pathways.

In vitro antitumor immune response induced by fusion of dendritic cells and Ewing's sarcoma cells

Human Ewing sarcoma A673 cells and human peripheral blood-derived DCs were fused to induce an antitumor activity against human EW. EW A673 cells and human peripheral blood-derived DCs were fused with polyethylene glycol (PEG). Mature DCs with highly expressed surface markers (CD80, CD86, CD83 and HLA-DR) were generated in vitro and flow cytometry. It showed that the highest fusion efficiency was 23.01%. T cell proliferation assay indicated that the novel dendritomas in fused DCs/A673 cells were the most potent in activation of autologous T cell proliferation. The IFN-gamma assay showed that The IFN-gamma secretion by CTLs activated by the novel dendritomas increased more than by other stimulators. CTL assay demonstrated that the novel dendritomas induced A673 cell-specific cytotoxic responses to lyse the A673 cells in the context of MHC class I. The data indicates that fusion of tumor cells with DCs is an attractive strategy to induce tumor rejection.

E1A Specifically Enhances Sensitivity to Topoisomerase IIα Targeting Anticancer Drug by Up-Regulating the Promoter Activity

DNA topoisomerases I and II (topo I and II) are nuclear enzymes involved in cellular replication and are targets for several anticancer drugs. We showed previously that E1A gene transfer enhanced the sensitivity of Ewing's sarcoma cells to the topo IIalpha targeting agents etoposide and Adriamycin in vitro and in vivo. To determine whether this effect was specific for topo IIalpha, we investigated the effect of E1A gene transfer on cell sensitivity to agents that target topo I and IIbeta. Transfecting TC71 human Ewing's sarcoma cells with an adenoviral vector containing the E1A gene enhanced their sensitivity to the topo IIalpha targeting agents etoposide (16-fold) and Adriamycin (8-fold). By contrast, E1A gene transfer did not affect cellular sensitivity to either amsacrine or camptothecin. Western blot analysis indicated that topo IIalpha protein levels increased 3.1-fold after E1A gene transfer, but topo I and IIbeta protein levels did not change. A plasmid containing topo IIalpha gene promoter with luciferase reporter gene was constructed to determine the effects of E1A gene transfer on the activity of the topo IIalpha promoter. E1A increased the activity of the topo IIalpha gene promoter by 3.5-fold relative to that of cells transfected with Ad-beta-gal. These results suggest that elevated topo IIalpha protein levels and enhanced sensitivity to topo IIalpha targeting agents were secondary to a direct effect of E1A on the topo IIalpha promoter. Combining E1A gene therapy with topo IIalpha targeting anticancer drugs may therefore have therapeutic benefit by increasing tumor cell sensitivity.

74. Specific Delivery of hTRAIL and Inhibition of Ewing's Sarcoma Growth by Transplantation of Viral Vector-Transduced Bone Marrow-Derived Mesenchymal Stem Cells (MSC)

Top of pageAbstract For the majority of pediatric patients with soft tissue tumors, some form of selective systemic therapy is required. Here we show that bone marrow-derived non-hematopoietic mesenchymal stem cells (MSCs) integrate into pediatrics soft tissue sarcoma xenografts as stromal fibroblasts. Importantly, exogenously administered MSC preferentially engraft at the sites of disseminated tumor nodules throughout the body. These findings suggest the development of novel anti-cancer therapies based on the local production of biological agents by gene manipulated MSC. We show that human MSC can be effectively transduced with adenovirus type 5-based vectors and with vectors based on adeno-associated virus (AAV). The majority of AAV serotypes tested (AAV1/6, 2, 3, 4, 5, and 8) are capable of transducing hMSC, albeit with relatively low efficiencies. Vector modifications to the AAV capsid, such as the inclusion of the RGD-tripeptide motif in capsids of either AAV1 or AAV2-based vectors, or modification of the vector genome to generate |[ldquo]|double-stranded|[rdquo]|, or |[ldquo]|self-complementary|[rdquo]| AAV vectors, both lead to significant increases in AAV-mediated hMSC gene transduction. Here, we examined whether hMSC producing human TNF-related apoptosis inducing ligand (hTRAIL) (hTRAIL-MSC) can inhibit the growth of subcutaneous human Ewing's Sarcoma xenografts in nude mice. MSC were transduced with an hTRAIL expressing adenovirus, or AAV vector. These hTRAIL-MSC produced hTRAIL and could directly induce apoptosis of A673 Ewing's sarcoma cells in co-culture experiments in vitro. When co-injected with A673 sarcoma cells into the flanks of nude mice (1:2 ratio of hMSC to A673 sarcoma cells), all treated animals failed to develop tumors (n=5). Control animals co-injected with eGFP-MSC (Ad-eGFP transduced hMSC) and A673 sarcoma cells (n=5), or A673 sarcoma cells alone (n=10) all developed tumors. We then injected hTRAIL-MSC intravenously (IV) (single dose of 5 |[times]| 105 MSC) into nude mice bearing established subcutaneous A673 tumors; tumor growth was inhibited approximately 30% as compared to untreated controls, and as compared to controls treated with eGFP-MSC (n=5) (p=0.01). Immunohistochemical analysis of hTRAIL-MSC treated animals showed engraftment and differentiation of hMSC into myofibroblast-like tumor-associated stroma cells. IV injected hTRAIL-MSC prolonged the survival of mice bearing A673 Ewing sarcoma grafts as compared to untreated animals, or control (eGFP-MSC) treated animals. These data suggest that IV administered gene-modified MSC can inhibit the growth of pediatric soft tissue sarcomas. Importantly, the antitumor effects were only achieved when MSC were integrated into the tumor microenvironment, but not when MSC were injected subcutaneously into the opposite flank. Therefore, tumor inhibition required spatial proximity of MSC to the malignant cells. These findings suggest that TRAIL can serve as a direct inhibitor of tumor proliferation and suggest the use of gene-manipulated MSC for pediatric cancer therapy.

A Small Interfering RNA Targeting Vascular Endothelial Growth Factor Inhibits Ewing's Sarcoma Growth in a Xenograft Mouse Model

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising therapeutic target for cancer. Vascular endothelial growth factor (VEGF) is a key regulator in vasculogenesis as well as in angiogenesis. TC71 human Ewing's sarcoma cells overexpress VEGF, with a shift in isoform production from membrane-bound VEGF189 to the more soluble VEGF165. Transfection of TC71 cells with a vector-based VEGF targeted small interfering RNA expression system (VEGFsi) inhibited VEGF165 expression by 80% and VEGF165 protein production by 98%, with no alteration in VEGF189 expression. Human microvascular endothelial cell proliferation and migration induced by conditioned medium from VEGFsi-transfected TC71 cells was significantly less than that induced by conditioned medium from TC71 cells and control vector-transfected TC71 cells. Furthermore, after s.c. injection into athymic nu/nu mice, the tumor growth of VEGFsi-expressing TC71 cells was significantly less than that of parental or control vector-transfected cells. Vessel density as assessed by CD31 immunohistochemical analysis and VEGF165 expression as assessed by Northern blotting were also decreased. Intratumor gene therapy with polyethylenimine/VEGFsi also resulted in tumor growth suppression. When inoculated into the tibias of nude mice, VEGFsi-expressing TC71 cells induced osteolytic bone lesions that were less severe than those induced by control groups. These data suggest that targeting VEGF165 may provide a therapeutic option for Ewing's sarcoma.

Herceptin Down-Regulates HER-2/neu and Vascular Endothelial Growth Factor Expression and Enhances Taxol-Induced Cytotoxicity of Human Ewing's Sarcoma Cells In vitro and In vivo

We have previously shown that high levels of HER-2/neu protein were overexpressed in human Ewing's sarcoma cells (TC71, SK-ES1) relative to normal human osteoblasts. The purpose of this study was to determine whether herceptin alone or in combination with chemotherapeutic agents could inhibit the growth of Ewing's sarcoma in vitro and in vivo. Western blot analysis showed that the protein levels of HER-2/neu were decreased following herceptin treatment. Cell growth was also inhibited by herceptin in a dose-dependent manner with an IC(50) of 4 mg/mL in TC71 and SK-ES1 cell line, whereas human immunoglobin had no effect. Northern blot and ELISA showed the RNA expression and protein levels of vascular endothelial growth factor were also inhibited by herceptin treatment with no alteration in HIF-1alpha protein and topoisomerase IIalpha expression. Furthermore, Ewing's sarcoma tumor growth was significantly delayed by 100 mg/kg herceptin treatment in our Ewing's sarcoma xenograft mouse model. Combining taxol with herceptin resulted in additive cytotoxicity, whereas herceptin-etoposide, doxorubicin, and 9-nitrocamptothecin combinations did not. Taxol-herceptin enhanced growth inhibition in TC71 cells in vitro compared with either agent alone. Ewing's sarcoma growth was also delayed in vivo and mean tumor size was significantly lower in mice treated with herceptin plus taxol than in those receiving taxol or herceptin alone. These data suggest that herceptin in combination with taxol may be a therapeutic option in the treatment of Ewing's sarcoma.

Antitumor effect of nutrient synergy on human osteosarcoma cells U-2OS, MNNG-HOS and Ewing's sarcoma SK-ES.1.

Current treatment of osteosarcoma is associated with poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new, safe and effective treatment strategies are needed. We investigated the effect of a unique mixture of nutrients containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (EGCG) on human osteosarcoma cell lines U-2OS, MNNG-HOS, and Ewing's sarcoma SK-ES-1 by measuring: cell proliferation, expression of matrix metalloproteinase-2 (MMP-2), MMP-9, and invasive and angiogenesis potential. Cell proliferation was evaluated by MTT assay, matrix metalloproteinases (MMP) expression by gelatinase zymography, VEGF expression by ELISA, and invasion through Matrigel. Cells were also treated with phorbol 12-myristate 13-acetate (PMA) to study enhanced MMP and VEGF expression. The invasion of osteosarcoma U-2OS and MNNG-HOS cells through Matrigel was significantly reduced in a dose-dependent fashion, with 100% inhibition of invasion of U-2OS cells at 100 microg/ml, and MNNG cells at 50 microg/ml concentration of the synergistically acting nutrient mixture. Ewing's sarcoma SK-ES-1 cells were not invasive. Nutrient synergy (NS) exhibited a dose response antiproliferative effect on osteosarcoma U-2OS cells, reaching 67% at 1000 microg/ml of NS; no significant suppression of cell proliferation was seen with MNNG or Ewing's sarcoma cells. Zymography showed dose-dependent inhibition of MMP secretion by all three cell lines in the presence of NS. VEGF secretion by U-2OS cells was completely blocked at 500 microg/ml of NS. Our results suggest NS is an excellent candidate for therapeutic use in the treatment of osteosarcoma, by inhibiting cancer cell invasion, and secretion of MMPs and VEGF, all critical parameters for cancer control and prevention.

Synthesis and DNA binding properties of novel benzo[b]isoquino[2,3-h]-naphthyridines

Several benzo[b]isoquino[2,3-h]-naphthyridines have been prepared via formal hetero-Diels Alder reaction of N-aryl imines as a key step. These compounds have different side chains at C-11, and a cis or trans configuration at the C-8a,C-14a ring junction. Binding constants for the interaction with oligonucleotides and polynucleotides were determined by UV absorption and melting experiments. NMR experiments (NOE) revealed that the cis isomers, showing a slightly folded structure, preferentially bind to the minor groove of AT-rich oligomers. In contrast, the trans isomers prefer the CG-rich sequences, leading to cap-complexes with the isoquinoline moiety stacked at the top of the double helix, in agreement with the flatter shape, and with a preference for the 3'-terminals, as found for camptothecins. Models of the complexes were built up by molecular dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Cytotoxicity assays against human Ewing sarcoma cell lines RD-ES and CAD-ES1 were performed.

The epidermal growth factor receptor HER2 is not a major therapeutic target in Ewing sarcoma.

Although chimeric EWS gene and Ets gene fusions are pathognomonic of Ewing sarcoma (ES) and primitive neuroectodermal tumors (PNET), the molecular pathogenesis of these pediatric malignancies is poorly understood. Recently, the human epidermal growth factor (HER)-2 receptor, which plays an important role in the biology of certain epithelial cancers, has been implicated in ES tumor cell line growth and chemosensitivity. To investigate whether HER2 might be a rational target for ES/PNET therapy, five ES cell lines and 13 archival primary ES/PNET samples were examined by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for evidence of HER2 overexpression. Although several ES cell lines demonstrated modest constitutive HER2 expression by immunoblot, none of the ES cell lines or primary tumor samples showed evidence of HER2 overexpression by IHC or HER2 gene amplification by FISH. Moreover, treatment of human ES cell lines with the HER2-targeted agent trastuzumab (Herceptin) had little effect on cell survival, proliferation, or growth in semi-solid medium. These results suggest that HER2 is not a biologically or therapeutically important pathway in ES/PNET.

Transformation induced by Ewing's sarcoma associated EWS/FLI-1 is suppressed by KRAB/FLI-1.

Ewing's sarcoma is a childhood bone tumour with poor prognosis, most commonly associated with a t(11;22)(q24;q12) reciprocal translocation that fuses the EWS and FLI-1 genes, resulting in the production of an aberrant chimeric transcription factor EWS/FLI-1. To elucidate the mechanisms by which EWS/FLI-1 mediates transformation in mouse models, we have generated a murine Ews/Fli-1 fusion protein. We demonstrate that this protein transforms fibroblast cells in vitro similar to human EWS/FLI-1 as demonstrated by serum and anchorage-independent growth, the formation of tumours in nude mice and elevation of the oncogenic marker c-myc. Furthermore, transformation of these cells was inhibited by a specific repressor, KRAB/FLI-1. The KRAB/FLI-1 repressor also suppressed the tumorigenic phenotype of a human Ewing's sarcoma cell line. These findings suggest that the transformed phenotype of Ewing's sarcoma cells can be reversed by using the sequence-specific FLI-1-DNA-binding domain to target a gene repressor domain. The inhibition of EWS/FLI-1 is the first demonstration of the KRAB domain suppressing the action of an ETS factor. This approach provides potential avenues for the elucidation of the biological mechanisms of EWS/FLI-1 oncogenesis and the development of novel therapeutic strategies.

Alteration of mesodermal cell differentiation by EWS/FLI-1, the oncogene implicated in Ewing's sarcoma.

The chimeric fusion gene EWS/FLI-1 is detected in most cases of Ewing's sarcoma (ES), the second most common malignant bone tumor of childhood. Although 80% of ES tumors develop in skeletal sites, the remainder can arise in almost any soft tissue location. The lineage of the cell developing the EWS/FLI-1 gene fusion has not been fully characterized but is generally considered to be of either mesenchymal or neural crest origin. To study this oncogene in a conceptually relevant target cell, EWS/FLI-1 was introduced into the murine cell line C2C12, a myoblast cell line capable of differentiation into muscle, bone, or fat. In this cellular context, EWS/FLI-1 profoundly inhibited the myogenic differentiation program. The block in C2C12 myogenic differentiation required the nuclear localization and DNA-binding functions of EWS/FLI-1 and was mediated by transcriptional and posttranscriptional suppression of the myogenic transcription factors MyoD and myogenin. Interestingly, C2C12-EWS/FLI-1 cells constitutively expressed alkaline phosphatase, a bone lineage marker, and were alkaline phosphatase positive by histochemistry but showed no other evidence of bone lineage commitment. Consistent with recent findings in human ES tumor cell lines, C2C12-EWS/FLI-1 cells constitutively expressed cyclin D1 and demonstrated decreased expression of the cell cycle regulator p21(cip1), even under differentiation conditions and at confluent density. This C2C12-EWS/FLI-1 cell model may assist in the identification of novel differentially expressed genes relevant to ES and provide further insight into the cell(s) of origin developing ES-associated genetic fusions.

Inhibition of proliferation in human MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma cell lines in vitro and in vivo by antagonists of growth hormone-releasing hormone: effects on insulin-like growth factor II.

Antagonists of growth hormone-releasing hormone (GH-RH) can inhibit the proliferation of various tumors either indirectly through the suppression of the pituitary growth hormone/hepatic insulin-like growth factor I (IGF-I) axis and the lowering of serum IGF-I concentration or directly by reducing the levels of IGF-I and IGF-II and their mRNA expression in tumors and blocking the effect of autocrine GH-RH. In this study, the authors investigated the effects of the GH-RH antagonist JV-1-38 on MNNG/HOS human osteosarcoma and SK-ES-1 human Ewing sarcoma cell lines. Male nude mice bearing subcutaneous xenografts of MNNG/HOS or SK-ES-1 tumors were treated subcutaneously with JV-1-38 at a dose of 20 microg twice daily for 4 weeks. The concentrations of IGF-I and IGF-II in serum and in tumor tissue were measured by radioimmunoassay. Tumor and liver levels of mRNA for IGF-I and IGF-II were determined by reverse transcriptase-polymerase chain reaction analysis. The effects of JV-1-38, IGF-I, and IGF-II on cell proliferation in vitro were evaluated. GH-RH antagonist significantly (P < 0.05) inhibited the tumor volume and tumor weight of MNNG/HOS and SK-ES-1 tumors by > 50% after 4 weeks and increased tumor doubling time. JV-1-38 lowered the serum IGF-I level, decreased the expression of mRNA for IGF-I in the liver, and significantly (P < 0.05-0.01) reduced the concentration of IGF-II and mRNA levels for IGF-II in both sarcomas. The concentration of IGF-I was lowered only in SK-ES-1 tumors. In vitro, the proliferation of SK-ES-1 and MNNG/HOS cells was inhibited by JV-1-38 and by antisera to IGF-I and IGF-II. The inhibition of MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma by GH-RH antagonists was linked to a suppression of IGF-II production in tumors. However, in SK-ES-1 tumors, the effects on IGF-I also may be involved.