Biomedical subjects

Carol J Thiele

Publications and source records attributed to Carol J Thiele.

At least 19 recordsLinked to original sources

PubMed2007

Credentialing preclinical pediatric xenograft models using gene expression and tissue microarray analysis.

Human tumor xenografts have been used extensively for rapid screening of the efficacy of anticancer drugs for the past 35 years. The selection of appropriate xenograft models for drug testing has been largely empirical and has not incorporated a similarity to the tumor type of origin at the molecular level. This study is the first comprehensive analysis of the transcriptome of a large set of pediatric xenografts, which are currently used for preclinical drug testing. Suitable models representing the tumor type of origin were identified. It was found that the characteristic expression patterns of the primary tumors were maintained in the corresponding xenografts for the majority of samples. Because a prerequisite for developing rationally designed drugs is that the target is expressed at the protein level, we developed tissue arrays from these xenografts and corroborated that high mRNA levels yielded high protein levels for two tested genes. The web database and availability of tissue arrays will allow for the rapid confirmation of the expression of potential targets at both the mRNA and the protein level for molecularly targeted agents. The database will facilitate the identification of tumor markers predictive of response to tested agents as well as the discovery of new molecular targets.

Cell Line, Tumor↗

PubMed2006

Molecular cloning and characterization of human Castor, a novel human gene upregulated during cell differentiation.

Castor is a zinc finger transcription factor that controls cell fate within neuroblast cell lineages in Drosophila melanogaster. Here, we describe the cloning and characterization of a human castor gene (CASZ1) that is structurally homologous to Drosophila castor. We find the expression of castor gene is increased when cells of neural origin as well as mesenchymal origin are induced to differentiation. CASZ1 is expressed in a number of normal tissues and exists in at least two mRNA species of 4.4 and 8.0kb. They are named hCasz5 and hCasz11 because the predicted proteins have 5 and 11 zinc fingers, respectively. Deletion analysis of the proximal 5'-flanking sequences delineates sequences sufficient to drive transcription in cells of neural and non-neural origin. Both hCasz5 and hCasz11 localize predominantly in the nucleus, consistent with their role as Zn-finger containing transcription factor. CASZ1 is expressed in a number of human tumors and localizes to a chromosomal region frequently lost in tumors of neuroectodermal origin.

Amino Acid Sequence↗

PubMed2006

Brain-derived neurotrophic factor activation of TrkB induces vascular endothelial growth factor expression via hypoxia-inducible factor-1alpha in neuroblastoma cells.

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Recently, we have shown that insulin-like growth factor-I and serum-derived growth factors stimulate VEGF expression in neuroblastoma cells via induction of hypoxia-inducible factor-1alpha (HIF-1alpha). Because another marker of poor prognosis in neuroblastoma tumors is high expression of brain-derived neurotrophic factor (BDNF) and its tyrosine kinase receptor, TrkB, we sought to evaluate the involvement of BDNF and TrkB in the regulation of VEGF expression. VEGF mRNA levels in neuroblastoma cells cultured in serum-free media increased after 8 to 16 hours in BDNF. BDNF induced increases in VEGF and HIF-1alpha protein, whereas HIF-1beta levels were unaffected. BDNF induced a 2- to 4-fold increase in VEGF promoter activity, which could be abrogated if the hypoxia response element in the VEGF promoter was mutated. Transfection of HIF-1alpha small interfering RNA blocked BDNF-stimulated increases in VEGF promoter activity and VEGF protein expression. The BDNF-stimulated increases in HIF-1alpha and VEGF expression required TrkB tyrosine kinase activity and were completely blocked by inhibitors of phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) pathways. These data indicate that BDNF plays a role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to BDNF/TrkB, PI3K, mTOR signal transduction pathways, and/or HIF-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.

Brain-Derived Neurotrophic Factor↗

PubMed2006

Targeting caspase 8 to reduce the formation of metastases in neuroblastoma.

The clinical challenge in neuroblastoma is the presence of metastasis at diagnosis in the majority of patients. Caspase 8 is an integral protein in death receptor-associated apoptosis, and loss of caspase 8 via the epigenetic phenomenon of methylation in neuroblastoma has led to increased resistance to chemotherapy. Recent evidence suggests that caspase 8 loss may also contribute to a metastatic phenotype; thus, caspase 8 may prove to be an attractive target for therapy both in treating primary tumours as well as preventing and treating metastatic lesions. Numerous methods have been described to manipulate caspase 8 levels both in vitro and in vivo, and investigation into caspase 8 isoforms may also bring forth additional therapeutic targets.

Animals↗

PubMed2005

TrkA induces apoptosis of neuroblastoma cells and does so via a p53-dependent mechanism.

Neuroblastoma (NB) is the most frequent solid extracranial tumor in children. Its clinical prognosis correlates with the expression of members of the Trk neurotrophin receptor family, which includes TrkA and TrkB. TrkA expression is associated with favorable prognosis, whereas TrkB expression is associated with poor prognosis. Here we show that TrkA expression induces the apoptosis of NB cells and does so by modulating the levels or activities of a number of proteins involved in regulating cell survival and apoptosis, including p53, Bcl-2, and caspase-3. TrkA increased the expression of p53 target proteins and failed to induce apoptosis in cells where p53 was inactivated by mutation or via expression of dominant inhibitory p53 or E1B55K, indicating that TrkA mediates apoptosis, at least in part, through p53. Treatment with a caspase inhibitor or overexpression of Bcl-X(L) also prevented TrkA from inducing apoptosis. In contrast, elevated expression of TrkA in non-transformed sympathetic neurons resulted in the suppression of p53 levels and enhanced survival. These results identify apoptosis as a novel biological response of TrkA in NB cells and imply that TrkA is a good prognosis marker for NB due in part to its ability to mediate apoptosis when expressed at sufficient levels.

Adenoviridae↗

PubMed2005

Topotecan blocks hypoxia-inducible factor-1alpha and vascular endothelial growth factor expression induced by insulin-like growth factor-I in neuroblastoma cells.

The extent of angiogenesis and/or vascular endothelial growth factor (VEGF) expression in neuroblastoma tumors correlates with metastases, N-myc amplification, and poor clinical outcome. Understanding the mechanisms regulating VEGF expression in neuroblastoma cells provides additional therapeutic options to control neuroblastoma tumor growth. VEGF mRNA is controlled by growth factors and hypoxia via the transcription factor hypoxia-inducible factor (HIF-1alpha). HIF-1alpha protein levels are regulated by the von Hippel Lindau tumor suppressor gene, VHL, which targets HIF-1alpha degradation. To determine whether the levels of VEGF in neuroblastomas are due to mutations in VHL, we evaluated genomic DNA from 15 neuroblastoma cell lines using PCR. We found no mutations in exons 1, 2, or 3 of the VHL gene. VEGF mRNA levels in neuroblastoma cells cultured in serum-free medium increased after 8 to 16 hours in serum, insulin-like growth factor-I (IGF-I), epidermal growth factor, or platelet-derived growth factor. Serum/IGF-I induced increases in HIF-1alpha protein that temporally paralleled increases in VEGF mRNA, whereas HIF-1beta levels were unaffected. VEGF and HIF-1alpha levels were blocked by inhibitors of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Furthermore, we confirmed that HIF-1alpha mediates approximately 40% of the growth factor activity stimulating VEGF protein expression. Topotecan blocked the IGF-I-stimulated increase in HIF-1alpha but not HIF-1beta, and this resulted in a decrease in VEGF in four neuroblastoma cell lines tested. These data indicate that growth factors in an autocrine or paracrine manner play a major role in regulating VEGF levels in neuroblastoma cells and that targeted therapies to phosphatidylinositol 3-kinase, mammalian target of rapamycin, and/or HIF-1alpha have the potential to inhibit VEGF expression and limit neuroblastoma tumor growth.

Antineoplastic Agents↗

PubMed2005

Genetic and pharmacologic identification of Akt as a mediator of brain-derived neurotrophic factor/TrkB rescue of neuroblastoma cells from chemotherapy-induced cell death.

Patients whose neuroblastoma tumors express high levels of brain-derived neurotrophic factor (BDNF) and TrkB have an unfavorable prognosis. Our previous studies indicated that BDNF activation of the TrkB signal transduction pathway blocked the cytotoxic effects of chemotherapeutic drugs via the phosphatidylinositol 3-kinase pathway. Akt is an important downstream target of phosphatidylinositol 3-kinase and functions to regulate cell survival, proliferation, and protein synthesis. In this study, we examined whether Akt is required and sufficient to mediate BDNF/TrkB protection of neuroblastoma cells from chemotherapy. Transient transfection of a constitutively active Akt (Akt-Myr) into TrkB-expressing SY5Y cells (TB8 cells) increases Akt activation and attenuates the cell death induced by chemotherapeutic reagents in the absence of BDNF. Furthermore, expression of a dominant-negative Akt (Akt-K179A) blocks the ability of BDNF to rescue TB8 cells from chemotherapy-induced cell death. Pharmacologic inhibition of Akt, with PIA6, a phosphatidylinositol ether lipid analogue (PIA), blocks BDNF-induced phosphorylation of Akt and the downstream target of Akt. PIA6 sensitizes neuroblastoma cells to chemotherapy and attenuates BDNF protection of neuroblastoma cells from chemotherapy-induced cell death. These results indicate that Akt is a key signaling component by which BDNF activation of the TrkB signal transduction pathway protects neuroblastoma cells from chemotherapy-induced cell death. This study raises the possibility that novel pharmacologic inhibitors of Akt may enhance the effectiveness of chemotherapeutic agents in the treatment of neuroblastoma tumors.

Brain-Derived Neurotrophic Factor↗

PubMed2005

HUlip, a human homologue of unc-33-like phosphoprotein of Caenorhabditis elegans; Immunohistochemical localization in the developing human brain and patterns of expression in nervous system tumors.

HUlip is a human homologue of a C. elegans gene, unc-33, that is developmentally regulated during maturation of the nervous system. HUlip is highly expressed only in the fetal brain and spinal cord, and is undetected in the adult brain. The purpose of this study was to investigate the pattern of hUlip expression in the developing human brain and nervous system tumors. Ten human brains at different developmental stages and 118 cases of nervous system tumor tissues were examined by immunohistochemistry. Twelve related tumor cell lines were also analyzed by northern blotting and immunoblotting. HUlip was expressed in late fetal and early postnatal brains; strongly in the neurons of the brain stem, basal ganglia/thalamus, and dentate gyrus of the hippocampus, and relatively weakly in the cerebral and cerebellar cortex. Among tumors, hUlip expression was easily detected in tumor cells undergoing neuronal differentiation such as ganglioneuroblastomas and ganglioneuromas. Furthermore, hUlip immunoreactivity was also found in various brain tumors showing neuronal differentiation: central neurocytomas (6 of 6 cases were positive), medulloblastomas (5/11), atypical teratoid rhabdoid tumor (1/1) and gangliogliomas (4/7). Some astrocytic tumors also showed weak positivity: astrocytomas (1 of 5 cases), anaplastic astrocytomas (2/5), and glioblastomas (3/11). Subependymal giant cell astrocytomas and subependymomas, which are of controversial histogenetic origin, showed strong hUlip immunoreactivity. The results of this study indicate that the expression of hUlip protein is distinctly restricted to the late fetal and early postnatal periods of human nervous system development and to certain subsets of nervous system tumors. The exact function of hUlip needs to be further clarified; yet the results of our study strongly imply that hUlip function is important in human nervous system development and its aberrant expression in various types of nervous system tumors suggests a role of hUlip as an oncofetal neural antigen.

Astrocytes↗

PubMed2005

Pediatric pancreatoblastoma: histopathologic and cytogenetic characterization of tumor and derived cell line.

Little is known of the molecular events underlying the genesis of pancreatoblastoma tumors in the pediatric population. Such studies have been limited by the rare nature of the disease, infrequent reports detailing cytogenetic alterations, and the lack of availability of cell lines for biologic studies. We present the isolation of a cell line from a 14-year-old boy with malignant pancreatoblastoma, and its cytogenetic characterization using spectral karyotyping and comparative genomic hybridization (CGH). The cytogenetic analysis revealed an exceedingly complex cytogenetic karyotype, with 33 aberrant chromosomes. CGH revealed multiple regions of chromosomal loss and gain, including a region on 8q gained in adult pancreatic cancers, one that frequently contains the MYC oncogene.

Adolescent↗

PubMed2004

Interferon gamma enhances the effectiveness of tumor necrosis factor-related apoptosis-inducing ligand receptor agonists in a xenograft model of Ewing's sarcoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptosis in a variety of tumors, including most cell lines derived from Ewing's sarcoma family of tumors, an aggressive sarcoma that afflicts children and young adults. To determine the in vivo efficacy of TRAIL receptor agonists in Ewing's sarcoma family of tumors, mice with orthotopic xenografts were treated with anti-TRAIL-R2 monoclonal antibody or TRAIL/Apo2L in a model that can identify effects on both primary tumors and metastases. Administration of either agonist slowed tumor growth in 60% of animals and induced durable remissions in 11 to 19% but did not alter the incidence of metastatic disease. Response rates were not improved by concurrent doxorubicin treatment. Cells recovered from both TRAIL receptor agonist-treated and nontreated tumors were found to be resistant to TRAIL-induced death in vitro unless pretreated with interferon (IFN) gamma. This resistance coincided with a selective down-regulation of TRAIL receptor expression on tumor cells. In vivo treatment with IFNgamma increased tumor expression of TRAIL receptors and caspase 8, but did not increase the antitumor effect of TRAIL receptor agonists on primary tumors. However, IFNgamma treatment alone or in combination with a TRAIL receptor agonist significantly decreased the incidence of metastatic disease and the combination of TRAIL receptor agonist therapy with IFNgamma-mediated impressive effects on both primary tumors and metastatic disease. These data demonstrate that in vivo growth favors TRAIL resistance but that TRAIL receptor agonists are active in Ewing's sarcoma family of tumors and that the combination of TRAIL receptor agonists with IFNgamma is a potent regimen in this disease capable of controlling both primary and metastatic tumors.

Animals↗

PubMed2004

Glycogen synthase kinase 3beta (GSK3beta) mediates 6-hydroxydopamine-induced neuronal death.

The causes of sporadic Parkinson's disease (PD) are poorly understood. 6-Hydroxydopamine (6-OHDA), a PD mimetic, is widely used to model this neurodegenerative disorder in vitro and in vivo; however, the underlying mechanisms remain incompletely elucidated. We demonstrate here that 6-OHDA evoked endoplasmic reticulum (ER) stress, which was characterized by an up-regulation in the expression of GRP78 and GADD153 (Chop), cleavage of procaspase-12, and phosphorylation of eukaryotic initiation factor-2 alpha in a human dopaminergic neuronal cell line (SH-SY5Y) and cultured rat cerebellar granule neurons (CGNs). Glycogen synthase kinase-3 beta (GSK3beta) responds to ER stress, and its activity is regulated by phosphorylation. 6-OHDA significantly inhibited phosphorylation of GSK3beta at Ser9, whereas it induced hyperphosphorylation of Tyr216 with little effect on GSK3beta expression in SH-SY5Y cells and PC12 cells (a rat dopamine cell line), as well as CGNs. Furthermore, 6-OHDA decreased the expression of cyclin D1, a substrate of GSK3beta, and dephosphorylated Akt, the upstream signaling component of GSK3beta. Protein phosphatase 2A (PP2A), an ER stress-responsive phosphatase, was involved in 6-OHDA-induced GSK3beta dephosphorylation (Ser9). Blocking GSK3beta activity by selective inhibitors (lithium, TDZD-8, and L803-mts) prevented 6-OHDA-induced cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), DNA fragmentations and cell death. With a tetracycline (Tet)-controlled TrkB inducible system, we demonstrated that activation of TrkB in SH-SY5Y cells alleviated 6-OHDA-induced GSK3beta dephosphorylation (Ser9) and ameliorated 6-OHDA neurotoxicity. TrkB activation also protected CGNs against 6-OHDA-induced damage. Although antioxidants also offered neuroprotection, they had little effect on 6-OHDA-induced GSK3beta activation. These results suggest that GSK3beta is a critical intermediate in pro-apoptotic signaling cascades that are associated with neurodegenerative diseases, thus providing a potential target site amenable to pharmacological intervention.

Animals↗

PubMed2004

NGF activation of TrkA decreases N-myc expression via MAPK path leading to a decrease in neuroblastoma cell number.

In neuroblastoma (NB), expression of the TrkA receptor is correlated with good prognosis while N-myc amplification is correlated with poor prognosis. Decreased N-myc levels are key to controlling growth and inducing differentiation in NB cells. In this report, we detail mechanisms by which nerve growth factor (NGF) decreases N-myc levels in TrkA-transfected NB cells and its effect on NB cell proliferation. NGF induced a decrease in N-myc mRNA within 1 h of treatment that occurred in the presence of cycloheximide. The stability of N-myc mRNA was not affected by NGF, indicating a transcriptional control of N-myc mRNA by NGF. NGF but not brain-derived neurotrophic factor (BDNF) decreased N-myc levels demonstrating that p75 alone was not involved. The NGF-induced decrease in N-myc expression was blocked by the Trk tyrosine kinase (TK) antagonist K252a indicating that signals transduced by Trk TK downstream targets were involved. Pharmacologic inhibitors implicated the mitogen-activated protein kinase (MAPK) path. This was supported by the finding that expression of a constitutively activated component of the MAPK path, MAPK kinase (MEK), decreased N-myc levels. Alterations in the level of N-myc are known to alter NB cell cycle progression by affecting the levels of E2Fs and p27(kip1). Consistent with these findings, NGF decreased NB cell number and decreased cyclin E-dependent kinase activity via an increase in p27(kip1). Thus, our results indicate that the MAP kinase is selectively involved in the NGF-induced N-myc downregulation through a transcriptional mechanism. Furthermore, NGF affects the time required for 15N TrkA cells to complete a replication cycle by decreasing N-myc, E2Fs, cyclin E kinase activity and increasing p27(kip1) binding to cyclin E kinase.

Carbazoles↗

PubMed2004

Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression.

BACKGROUND: Alternative treatment options are needed for advanced neuroblastoma patients because their prognosis remains poor after intensive chemotherapy. Neuroblastoma cells express platelet-derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the tyrosine kinase activities of c-Kit and PDGFR, on the growth of neuroblastoma cells in vivo and in vitro. METHODS: We tested seven human neuroblastoma cell lines for their sensitivity to imatinib. Cell viability was assessed by trypan blue dye exclusion. Apoptosis was evaluated by nuclear staining, flow cytometry, and western blotting. Protein assays included immunoprecipitation, western blotting, enzyme-linked immunosorbent assays, and immunohistochemistry. mRNA expression was assessed by northern blotting. We used a xenograft model in SCID mice (10 mice per group) to evaluate the effects of imatinib oral therapy (50 or 100 mg/kg every 12 hours for 14 days) on neuroblastoma tumor growth. All statistical tests were two-sided. RESULTS: All seven neuroblastoma cell lines treated with imatinib displayed concentration-dependent decreases in cell viability, which coincided with an induction of apoptosis, and with ligand-stimulated phosphorylation of c-Kit and PDGFR. The imatinib concentrations that caused 50% inhibition of growth and 50% inhibition of ligand-induced phosphorylation of these receptors were 9-13 micro M and 0.1-0.5 microM, respectively. Expression of VEGF, but not phosphorylation of Flk-1, its receptor, was reduced in neuroblastoma cells treated with imatinib at 10 microM or higher. Mice treated with imatinib at 50 mg/kg or 100 mg/kg had statistically significantly smaller tumors than control mice treated with vehicle (mean tumor volume in mice treated with imatinib at 50 mg/kg = 1546 mm3, in control mice = 2954 mm3; difference = 1408 mm3, 95% confidence interval [CI] = 657 to 2159 mm3; P<.001; mean tumor volume in mice treated with imatinib at 100 mg/kg = 463 mm3; difference = 2491 mm3, 95% CI = 1740 to 3242 mm3; P<.001). CONCLUSIONS: Imatinib inhibited the growth of neuroblastoma cells in vitro and in vivo. This inhibition was associated with suppression of PDGFR and c-Kit phosphorylation and inhibition of VEGF expression.

Administration, Oral↗

PubMed2004

Fibroblast growth factor 2 induces differentiation and apoptosis of Askin tumour cells.

Peripheral primitive neuroectodermal tumour (PNET)/Ewing's sarcoma (ES) and neuroblastoma (NB) are related tumours of neural crest origin with primitive neural characteristics. Fibroblast growth factor 2 (FGF2) is a critical signalling molecule for primitive neural crest cells. The treatment of NB cells with FGF2 variably affects biological characteristics such as growth and differentiation, while in PNET/ES, FGF2 predominantly induces apoptosis. The JK-GMS Askin tumour cell line can be induced to differentiate upon treatment with nerve growth factor (NGF), indicating the integrity of the cellular machinery necessary for differentiation. The present study assesses whether FGF2 can induce differentiation in JK-GMS cells. JK-GMS cells expressed high-affinity FGF receptors (FGFRs), and treatment with FGF2 induced phosphorylation of FGFR1 together with activation of extracellular signal-regulated kinases (ERK1/ERK2) and c-Jun N-terminal kinase (JNK). Subsequent biological effects were growth inhibition, neuronal differentiation, and apoptosis, and these changes were associated with increased expression of neurofilaments, reduction of c-myc and bcl-2 expression, and activation of caspase 3. Treatment of the cells with a specific inhibitor of the MAPK/extracellular signal-regulated kinase (MEK)-1, PD98059, predominantly inhibited the effects of FGF2 on growth, differentiation, and apoptosis, while an inhibitor of JNK reduced apoptosis, indicating that the ERK1/2 and JNK pathways are critical components of FGF2-mediated effects in JK-GMS cells. Additional comparative analyses of FGF2-mediated effects in two ES cell lines (CADO-ES, RD-ES) and a PNET cell line (SK-N-MC) showed pronounced differentiation in SK-N-MC, but not in CADO-ES or RD-ES cells. This study demonstrates that FGF2 can induce neuronal differentiation of PNET including Askin tumour. These findings clearly indicate that the FGF2-mediated signalling pathway plays a critical role in controlling the major properties of PNET cells and may provide a potential therapeutic target for PNET.

Apoptosis↗

PubMed2003

Targeting the tumor necrosis factor-related apoptosis-inducing ligand path in neuroblastoma.

The identification of the tumor necrosis factor (TNF) superfamily member TNF-related apoptosis-inducing ligand (TRAIL) a few years ago generated considerable enthusiasm for it as a potential cancer therapeutic agent. This is because TRAIL shows potent apoptosis inducing activity in a wide spectrum of transformed cell lines but not in cell lines derived from normal tissue origin. As the details in the signal transduction pathway of TRAIL-induced apoptosis are clarified, various defects of TRAIL pathway have been identified in TRAIL resistant cancer cells. Neuroblastoma is the most common extracranial solid tumor in children and those with a poor prognosis require more sensitive therapies. Unlike other cancer cells, most neuroblastoma cell lines are resistant to TRAIL induced apoptosis and the resistance correlates with caspase 8 deficiency, which is attributed to the methylation of the gene. Interferon (IFN)-gamma induces caspase 8 expression in most neuroblastoma cell lines regardless of the methylation status but fails to sensitize most NB to TRAIL. Further analysis indicates a TRAIL receptor deficiency contributes to TRAIL resistance in NB. Multiple lesions suggest that this path may play an important role in tumorigenesis and/ or evasion from therapies. Furthermore it indicates that the clinical application of TRAIL in NB will require a multi-modality approach. Important questions remain unanswered: How does IFN-gamma induce caspase 8 and why is the induction heterogeneous? How to stimulate the caspase 8 induction in cells that fail to respond to IFN-gamma? How to target other TRAIL pathway lesions with the clinically feasible approaches?

Apoptosis↗

PubMed2003

Cisplatin-induced cytotoxicity is blocked by brain-derived neurotrophic factor activation of TrkB signal transduction path in neuroblastoma.

We evaluated the ability of brain-derived neurotrophic factor (BDNF) to decrease the chemosensitivity of neuroblastoma cells to cisplatin. Two cell lines, one derived from SH-SY5Y (SY5Y-TB8) and the other from SK-N-AS (AS-TB8), transfected with a TrkB plasmid were generated, and used to assess the effects of activation of the TrkB signal transduction path on cisplatin (Cis) induced apoptosis. BDNF treatment of each of the TrkB expressing cells blocked cisplatin-induced cell death. BDNF's ability to rescue the cells from cisplatin-induced cell death was inhibited by treatment with the Trk tyrosine kinase inhibitor, K252a, and the phosphatidylinositol 3'-kinase (PI)-3-kinase inhibitor, LY294002. This indicates that the activation of the TrkB path through PI-3-kinase is required for BDNF's survival-promoting effects.

Antineoplastic Agents↗

PubMed2003

Induction of caspase 8 by interferon gamma renders some neuroblastoma (NB) cells sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but reveals that a lack of membrane TR1/TR2 also contributes to TRAIL resistance in NB.

The resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been attributed to a lack of caspase 8 expression. Here we demonstrate a clinically applicable molecular targeting strategy that not only increases caspase 8 expression ex vivo in NB cell lines but also in the tumor tissues of NB patients receiving IFN-gamma treatment. We identify the functional caspase 8 promoter, which is different from the methylated region reported previously, and show promoter activity is up-regulated by IFN-gamma through a IFN-gamma activation site-containing region. IFN-gamma also induces TRAIL expression in NB cell lines. However, the IFN-gamma restoration of caspase 8 in some NB cells revealed persistent TRAIL resistance in most NB cell lines examined. This additional lesion in the TRAIL path is because of a loss of cell membrane TRAIL receptors (TR1/TR2) not only in cell lines but in most of the NB tumor tissues evaluated. Restoration of TR2 expression by transfection enhances IFN-gamma-induced TRAIL sensitivity. Furthermore, we have found that we can improve TRAIL sensitivity in NB by reconstituting caspase 8 with IFN-gamma and TR2 with chemotherapeutic agents.

Antineoplastic Agents↗

PubMed2002

Potential use of imatinib in Ewing's Sarcoma: evidence for in vitro and in vivo activity.

BACKGROUND: Ewing's sarcoma cells express c-kit, a receptor tyrosine kinase, and its ligand, stem cell factor (SCF), creating a potential autocrine loop that may promote tumor survival. We thus examined whether the specific tyrosine kinase inhibitor imatinib mesylate (hereafter imatinib; formerly STI571) could inhibit the proliferation of Ewing's sarcoma cells in vitro and in vivo. METHODS: The effect of imatinib on c-kit expression and phosphorylation in Ewing's sarcoma cells was examined by immunoblotting. The effect of imatinib on cell growth and apoptosis was examined with an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and with a morphologic test and Annexin V staining, respectively. The effect of imatinib oral therapy (every 12 hours for 5-7 days) on primary tumor growth was assessed in Ewing's sarcoma xenografts in SCID/bg mice (5 or 10 mice per group). RESULTS: All Ewing's sarcoma cell lines tested were sensitive to imatinib-mediated apoptosis with a concentration inhibiting growth by 50% (IC50) of 10-12 micro M. Imatinib inhibited SCF-mediated c-kit phosphorylation (IC50 = 0.1-0.5 microM). In the xenograft model, imatinib treatment resulted in the regression or control of primary Ewing's sarcomas. After 6 days of treatment, the mean lower extremity volume including xenograft tumor was 3744 mm3 (95% confidence interval [CI] = 3050 to 4437 mm3), 1442 mm3 (95% CI = 931 to 1758 mm3), and 346 mm3 (95% CI = 131 to 622 mm3) in mice treated with carrier alone or with imatinib at 50 mg/kg or at 100 mg/kg, respectively. CONCLUSIONS: Imatinib interferes with growth of all Ewing's sarcoma cell lines tested in vitro and in vivo. Targeted inhibition of tyrosine kinase-dependent autocrine loops, therefore, may be a viable therapeutic strategy for Ewing's sarcoma.

Animals↗