Receptors In Neuroblastoma Research Articles

Feasibility and Therapeutic Potential of Combined Peptide Receptor Radionuclide Therapy With Intensive Chemotherapy for Pediatric Patients With Relapsed or Refractory Metastatic Neuroblastoma.

Recent evidence has demonstrated high expression of somatostatin receptors in neuroblastoma (NB) cells. Because of this, we endeavored to evaluate the diagnostic performance and clinical efficacy of 68Ga-DOTATATE PET/CT and peptide receptor radionuclide therapy (PRRT) using 177Lu-DOTATATE combined with chemotherapy in pediatric NB patients. In total, 14 pediatric patients with histopathologically confirmed NB underwent 68Ga-DOTATATE PET/CT. Among them, the patients who were refractory or relapsed after therapy with 131I-MIBG and had intensive uptake of 68Ga-DOTATATE were referred for PRRT using 177Lu-DOTATATE. Treatment response based on follow-up imaging was classified into complete response, partial response, stable disease, and progressive disease. After each cycle of PRRT, laboratory tests were performed for evaluation of hematological, renal, and hepatic toxicities. The CTCAE (Common Terminology Criteria for Adverse Events; version 4.03) was used for grading adverse event. Curie score and International Society of Pediatric Oncology Europe Neuroblastoma score were used for semiquantitative analysis of scans of patients who underwent PRRT. In addition, overall survival was calculated as the time interval between the date of the first cycle and the end of follow-up or death. Overall, 14 refractory NB children including 7 boys and 7 girls with a median age of 5.5 years (ranged from 4 to 9) underwent 68Ga-DOTATATE PET/CT. PET/CT was positive in 10/14 patients (71.4%), and the median number of detected lesions in positive patients was 2 (range, 1-13). Of 14 patients, 5 patients underwent PRRT, including 3 boys and 2 girls. A total of 19 PRRT cycles and 66.4 GBq 177Lu-DOTATATE were given. Among these 5 patients, 2 showed an initial complete response, which relapsed a few months later, 1 showed a partial response, and 2 showed progressive disease. According to the Kaplan-Meier test, the overall survival was estimated at 14.5 months (95% confidence interval, 8.9-20.1). In evaluation of PRRT-related toxicity according to the CTCAE, 4 patients showed grade 1, and 1 showed grade 2 leukopenia. Two patients showed grade 1, and 2 others showed grade 2 anemia. Two patients showed grade 1, and 3 patients showed grade 2 thrombocytopenia. Serum creatinine in 1 patient increased to grade 1. Combination of 177Lu-DOTATATE with chemotherapeutic agents might achieve worthwhile responses with low toxicity, encouraging survival in NB patients who have relapsed or are refractory to conventional therapy, including 131I-MIBG therapy. Imaging with 68Ga-DOTATATE PET/CT in such patients has a relatively high detection efficacy, demonstrating its potential use as an alternative imaging tool to conventional modalities such as 123I/131I-MIBG. However, further well-designed trials are highly warranted.

Parathyroid hormone-like hormone plays a dual role in neuroblastoma depending on PTH1R expression.

We have previously reported the expression of parathyroid hormone‐like hormone (PTHLH) in well‐differentiated, Schwannian stroma‐rich neuroblastic tumors. The aim of this study was to functionally assess the role of PTHLH and its receptor, PTH1R, in neuroblastoma. Stable knockdown of PTHLH and PTH1R was conducted in neuroblastoma cell lines to investigate the succeeding phenotype induced both in vitro and in vivo. Downregulation of PTHLH reduced MYCN expression and subsequently induced cell cycle arrest, senescence, and migration and invasion impairment in a MYCN‐amplified, TP53‐mutated neuroblastoma cell line. These phenotypes were associated with reduced tumorigenicity in a murine model. We also show that PTHLH expression is not under the control of the calcium‐sensing receptor in neuroblastoma. Conversely, its production is stimulated by epidermal growth factor receptor (EGFR). Accordingly, irreversible EGFR inhibition with canertinib abolished PTHLH expression. The oncogenic role of PTHLH appeared to be a consequence of its intracrine function, as downregulation of its receptor, PTH1R, increased anchorage‐independent growth and induced a more undifferentiated, invasive phenotype. Respectively, high PTH1R mRNA expression was found in MYCN nonamplified primary tumors and also significantly associated with other prognostic factors of good outcome. This study provides the first evidence of the dual role of PTHLH in the behavior of neuroblastomas. Moreover, the identification of EGFR as a transcriptional regulator of PTHLH in neuroblastoma provides a novel therapeutic opportunity to promote a less aggressive tumor phenotype through irreversible inhibition of EGFR tyrosine kinase activity.

Targeting tachykinin receptors in neuroblastoma.

Neuroblastoma is the most common extracranial tumor in children. Despite aggressive multimodal treatment, high-risk neuroblastoma remains a clinical challenge with survival rates below 50%. Adding targeted drugs to first-line therapy regimens is a promising approach to improve survival in these patients. TACR1 activation by substance P has been reported to be mitogenic in cancer cell lines. Tachykinin receptor (TACR1) antagonists are approved for clinical use as an antiemetic remedy since 2003. Tachykinin receptor inhibition has recently been shown to effectively reduce growth of several tumor types. Here, we report that neuroblastoma cell lines express TACR1, and that targeting TACR1 activity significantly reduced cell viability and induced apoptosis in neuroblastoma cell lines. Gene expression profiling revealed that TACR1 inhibition repressed E2F2 and induced TP53 signaling. Treating mice harboring established neuroblastoma xenograft tumors with Aprepitant also significantly reduced tumor burden. Thus, we provide evidence that the targeted inhibition of tachykinin receptor signaling shows therapeutic efficacy in preclinical models for high-risk neuroblastoma.

Abstract 3279: Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis

Abstract Chemerin is an adipokine and immunomodulating factor that promotes chemotaxis of immature dendritic cells, natural killer cells, macrophages and endothelial cells. Secreted as prochemerin with low activity, it can be C-terminally processed by different proteases expressed by a broad range of cell types and tissues. The resulting isoforms vary in receptor affinity and biological activity and are natural ligands for the G protein-coupled receptors (GPCRs) CMKLR1, GPR1 and CCLR2. To date, the activation of CMKLR1 (chemokine-like receptor 1) by chemerin and its role in metabolism and metabolic disorders as well as inflammation is best understood. Neuroblastoma (NB) is a malignancy of the sympathetic nervous system and the most common extracranial solid pediatric tumor. Several chemoattractant GPCRs have been suggested to promote tumor progression, angiogenesis and metastasis in NB. Although for some cancers a potential function has been suggested, the role of chemerin and its receptors in the NB tumor microenvironment remains unknown. In our study, the screening of microarray databases and analysis of neuroblastoma expression data showed a correlation between high CMKLR1, GPR1 and CCLR2 expression and a reduction in the overall survival probability. Expression of CMKLR1, GPR1, and chemerin was shown in nine neuroblastoma cell lines using RT-PCR, Western blot and immunocytochemistry. Furthermore, chemerin and CMKLR1 were also detected in neuroblastoma tumor tissue by immunohistochemistry. Stimulation of NB cell lines with active chemerin induced calcium mobilization and increased phosphorylation of MEK1/2 and ERK1/2 indicating an activation of the MAPK signaling pathway. Furthermore, chemerin stimulation led to increased NF-κB phosphorylation and translocation to the nucleus. The induction of NF-κB mediated signaling was observed by luciferase reporter assay. TNFα, IL-1β or serum stimulation increased chemerin protein expression and secretion in neuroblastoma cells. To assess the functional significance of chemerin and its receptors in neuroblastoma tumorigenesis, cell clones overexpressing or silenced for CMKLR1/ Chemerin/ GPR1 are used in NB animal models. Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Per Kogner, John Inge Johnsen, Baldur Sveinbjørnsson. Expression of chemerin and chemerin receptors in neuroblastoma: implications in tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3279. doi:10.1158/1538-7445.AM2015-3279

Mesenchymal stromal cell secretome up-regulates 47 kDa CXCR4 expression, and induce invasiveness in neuroblastoma cell lines.

Neuroblastoma accounts for 15% of childhood cancer deaths and presents with metastatic disease of the bone and the bone marrow at diagnosis in 70% of the cases. Previous studies have shown that the Mesenchymal Stromal Cell (MSC) secretome, triggers metastases in several cancer types such as breast and prostate cancer, but the specific role of the MSC factors in neuroblastoma metastasis is unclear. To better understand the effect of MSC secretome on chemokine receptors in neuroblastoma, and its role in metastasis, we studied a panel of 20 neuroblastoma cell lines, and compared their invasive potential towards MSC-conditioned-RPMI (mRPMI) and their cytokine receptor expression profiles. Western blot analysis revealed the expression of multiple CXCR4 isoforms in neuroblastoma cells. Among the five major isoforms, the expression of the 47 kDa isoform showed significant correlation with high invasiveness. Pretreatment with mRPMI up-regulated the expression of the 47 kDa CXCR4 isoform and also increased MMP-9 secretion, expression of integrin α3 and integrin β1, and the invasive potential of the cell; while blocking CXCR4 either with AMD 3100, a CXCR4 antagonist, or with an anti-47 kDa CXCR4 neutralizing antibody decreased the secretion of MMP-9, the expression of integrin α3 and integrin β1, and the invasive potential of the cell. Pretreatment with mRPMI also protected the 47 kDa CXCR4 isoform from ubiquitination and subsequent degradation. Our data suggest a modulatory role of the MSC secretome on the expression of the 47 kDa CXCR4 isoform and invasion potential of the neuroblastoma cells to the bone marrow.

Inhibition of Mer and Axl receptor tyrosine kinases leads to increased apoptosis and improved chemosensitivity in human neuroblastoma

Ectopic expression of Mer and Axl receptor tyrosine kinases (RTKs) are frequently found in various cancers as known to promote oncogenesis by activating antiapoptotic signaling pathways. However, the roles of these receptors in neuroblastoma remain unclear. We found Mer and Axl was co-expressed in neuroblastoma patient samples and cell lines. Ligand-dependent Mer or Axl activation led to an increase in phosphorylated ERK1/2, AKT and FAK indicating roles for these RTKs in multiple oncogenic processes. Furthermore, Mer and Axl knockdown led to apoptosis and inhibition of migration as well as a significant increase in chemosensitivity in response to cisplatin and vincristine treatment. Taken together, our results demonstrated that inhibition of Mer and Axl improved apoptotic response and chemosensitivity in neuroblastoma, providing new insights into development of novel therapeutic strategies by targeting these oncogenes.

Abstract 3990: Chemerin and chemerin receptors in neuroblastoma tumor microenvironment

Abstract Tumor promoting inflammatory cells as well as inflammatory mediators such as chemokines are important contributors to the tumor microenvironment as they can support tumor progression, angiogenesis and metastasis. Chemerin (also known as TIG-2 or RARRES2) is a chemoattractant factor for macrophages, immature DCs and NK-cells and a known adipokine involved in inflammation, metabolism and adipogenesis. Synthesized as a 163aa preproprotein, chemerin is N-terminally cleaved and secreted as inactive prochemerin. Following secretion, chemerin can be cleaved at the C-terminus by a variety of extracellular proteases resulting in several isoforms with varying length and biological activity. During inflammation initiation, maintenance and resolution the different chemerin isoforms may function pro- and/ or anti- inflammatory. Chemerin is a natural ligand for the G protein- coupled receptors CMKLR1 (Chem23) and GPR1. The role of chemerin and CMKLR1 in the tumor microenvironment has not been extensively studied. The aim of this work is to study the function of chemerin and its receptors in neuroblastoma (NB). Screening of mRNA expression arrays showed a correlation between high expression of CMKLR1 and GPR1 and a worsened prognosis in NB. Chemerin, CMKLR1 and GPR1 expression was detected in different neuroblastoma cell lines by RT-PCR, immunocytochemistry and Western Blot. Stimulation with chemerin resulted in rapid and transient ERK1/2 and Akt phosphorylation. TNF-α and IL-1β treatment increased chemerin and CMKLR1 protein levels. The functional significance of chemerin/ CMKLR1 in NB will be assessed by the use of NB animal models using cell clones silenced for chemerin/ CMKLR1. Citation Format: Conny Tuemmler, Igor Snapkov, Ugo L. Moens, Baldur Sveinbjørnsson. Chemerin and chemerin receptors in neuroblastoma tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2014-3990

Abstract SY25-03: Host and tumor genetic architecture influence clinically relevant oncogenic pathways in neuroblastoma

Abstract Cancer research is at a critical crossroads as genomic technologies have enabled potentially paradigm-shifting discoveries in host genetic alterations that lead to cancer, as well as the major oncogenic drivers that somatically arise and shape the ultimate tumor phenotype. Approaches such as genome-wide association studies (GWAS) to discover heritable susceptibility variants have largely occurred in parallel with tumor profiling efforts designed to discover somatically acquired mutations. We see tremendous opportunity in integrating these efforts under the broad hypothesis that the host's genetic blueprint not only influences susceptibility to cancer, but also significantly influences the cancer phenotype at diagnosis and the pathways upon which the cancer is dependent. By focusing our efforts on the pediatric cancer neuroblastoma, we are directly addressing an important childhood malignancy that continues to exact disproportionate morbidity and mortality. However, we have also shown that this disease also serves as a remarkable model for the study of human cancers in general, and we think that lessons learned here are likely to be more broadly applicable as we are addressing the concept that somatic mutations alone will not define the entire spectrum of oncogenic vulnerabilities in cancer cells. Until recently, the etiology of neuroblastoma was unknown. By studying the 1% of neuroblastoma cases that occur within families, we have discovered ALK and PHOX2B as the two major hereditary predisposition genes.1,2 In parallel, by studying the 99% of cases that occur sporadically in our ongoing GWAS, for which we have now analyzed 4,000 cases and 10,000 controls, we have shown that normal genetic variation at multiple loci influences neuroblastoma susceptibility.3-7 We have several other recently validated associations that are not yet published, including replicated signals in LIN28B and HACE1. Significant lessons from the GWAS to date include: 1) Neuroblastoma is a genetic disease; 2) every signal identified to date is intragenic; 3) associations are enriched in phenotypic subsets; and 4) many of the genes identified by the neuroblastoma GWAS not only influence malignant transformation of a developing neuroblastic cell, but also contribute to the maintenance of the malignant phenotype. For example, SNP variations in the LMO1 gene are highly associated with overexpression of this transcriptional co-factor, and genetic manipulation of expression shows that it functions as an oncogenic driver in a genotype specific manner.7 In other words, our data suggest that subjects harboring LMO1 risk alleles are much more likely to have tumors dependent on LMO1-influenced transcriptional regulation suggesting that oncogene addiction is also hardwired in the heritable genome. In parallel, we and others have shown that somatic activating mutations in the ALK kinase domain occur in ∼10% of sporadic cases.2,8-10 To seek other potential somatically acquired mutations that may be therapeutically relevant in the neuroblastoma coding genome, we have resequenced 99 high-risk neuroblastoma cases using a combination of whole exome, genome and transcriptome sequencing. Tumors contained a median 0.56 non-silent mutations per megabase of coding DNA, one of the lowest rates reported in cancer to date. ALK was the most commonly mutated gene (9%), and no other gene showed mutation frequency rates >5%. These data clearly indicate that somatic mutation in neuroblastoma is relatively rare, leading to the concept that other mechanisms, such as host polymorphic variation or germline mutations in key regulatory regions, play an important role in tumorigenesis. Ongoing work to be discussed at the symposium include efforts to identify and validate rare variants in the host genome that impact neuroblastoma susceptibility, understanding the functional relevance of DNA variation in the developing neuroblastoma and in tumor tissues, completion of our cataloguing of somatic mutations in high-risk disease, and efforts to understand the impact of non-coding DNA alterations in the host in tumor genomes on neuroblastoma clinical phenotype. Efforts to translate these efforts into deliverables for the clinic will be emphasized.

Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma

Neuroblastoma, a tumour derived from the peripheral sympathetic nervous system, is one of the most frequent solid tumours in childhood. It usually occurs sporadically but familial cases are observed, with a subset of cases occurring in association with congenital malformations of the neural crest being linked to germline mutations of the PHOX2B gene. Here we conducted genome-wide comparative genomic hybridization analysis on a large series of neuroblastomas. Copy number increase at the locus encoding the anaplastic lymphoma kinase (ALK) tyrosine kinase receptor was observed recurrently. One particularly informative case presented a high-level gene amplification that was strictly limited to ALK, indicating that this gene may contribute on its own to neuroblastoma development. Through subsequent direct sequencing of cell lines and primary tumour DNAs we identified somatic mutations of the ALK kinase domain that mainly clustered in two hotspots. Germline mutations were observed in two neuroblastoma families, indicating that ALK is a neuroblastoma predisposition gene. Mutated ALK proteins were overexpressed, hyperphosphorylated and showed constitutive kinase activity. The knockdown of ALK expression in ALK-mutated cells, but also in cell lines overexpressing a wild-type ALK, led to a marked decrease of cell proliferation. Altogether, these data identify ALK as a critical player in neuroblastoma development that may hence represent a very attractive therapeutic target in this disease that is still frequently fatal with current treatments.

Tumor cell‐associated neuropilin‐1 and vascular endothelial growth factor expression as determinants of tumor growth in neuroblastoma

We sought to characterize the expression of vascular endothelial growth factor (VEGF) and its receptors in neuroblastoma (NBL) and to correlate the results with N-myc (MYCN) expression and in vivo growth of these tumors. Two representative human-derived NBL cell lines, SK-N-AS (AS) with low and SK-N-DZ (DZ) with a high MYCN copy number, were used for the study. We examined their proliferation, VEGF and VEGF receptor expression in vitro and xenograft tumor growth in vivo. In parallel, human NBL specimens were analyzed for expression of VEGF and neuropilin-1 (NRP-1). DZ cells exhibited a 4-fold higher proliferation rate than AS. In contrast, VEGF protein expression was significantly higher in AS cells. NRP-1 was the only VEGF receptor produced in AS and DZ cells in vitro and in vivo. Both AS and DZ cells formed tumors in athymic mice but AS tumors grew 3.5 times larger than DZ tumors and had larger diameter tumor vessels. VEGF and NRP-1 expression was also demonstrated in human NBL specimens. Our studies indicate that VEGF and VEGF receptor expression in NBL tumor cells are associated with tumor growth and that angiogenic factors may serve as a biological marker together with already established MYCN amplification.

Study on the inhibition of protein production by L-cysteine derivatives of nucleic acid bases.

Isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acids bases were prepared as antisense compounds. In previous studies, we investigated the properties of these compounds in vitro, and revealed that these compounds in vivo regulated the cell death presumably due to the inhibition of protein production. In this study, western and northern blots were carried out in order to reveal the mechanism of this inhibition for N-methyl-D-aspartate receptor in neuroblastoma x glioma hybrid NG108-15 cell line. In addition, we investigated the resistance of these compounds against cell extract and the metabolism. In conclusion, we proved that these compounds inhibited the protein production by antisense mechanism.

5-Hydroxytryptamine 4(a) Receptor Is Coupled to the Gα Subunit of Heterotrimeric G13 Protein

Serotonin (5-hydroxytryptamine (5-HT)) is an important neurotransmitter that regulates multiple events in the central nervous system. Many of the 5-HT functions are mediated via G protein-coupled receptors that are coupled to multiple heterotrimeric G proteins, including G(s), G(i), and G(q) subfamilies (Martin, G. R., Eglen, R. M., Hamblin, M. W., Hoyer, D., and Yocca, F. (1998) Trends Pharmacol. Sci. 19, 2-4). Here we show for the first time that the 5-hydroxytryptamine 4(a) receptor (5-HT(4(a))) is coupled not only to heterotrimeric G(s) but also to G(13) protein, as assessed both by biochemical and functional assays. Using reconstitution of 5-HT(4(a)) receptor with different G proteins in Spodoptera frugiperda (Sf.9) cells, we have proved that agonist stimulation of receptor-induced guanosine 5'-(3-O-thio)triphosphate binding to Galpha(13) protein. We then determined that expression of 5-HT(4(a)) receptor in mammalian cells induced constitutive- as well as agonist-promoted activation of a transcription factor, serum response element, through the activation of Galpha(13) and RhoA. Finally, we have determined that expression of 5-HT(4(a)) receptor in neuroblastoma x glioma NIE-115 cells cause RhoA-dependent neurite retraction and cell rounding under basal conditions and after agonist stimulation. These data suggest that by activating 5-HT(4(a)) receptor-G(13) pathway, serotonin plays a prominent role in regulating neuronal architecture in addition to its classical role in neurotransmission.

Expression and functional analysis of the anaplastic lymphoma kinase (ALK) gene in tumor cell lines.

The initial identification of the ALK gene, expressed as C-terminal part of the transforming fusion protein NPM-ALK in the t(2;5)(p23;q35) lymphoma-associated chromosomal translocation, revealed a novel receptor tyrosine kinase (RTK). In order to expand the knowledge on ALK expression in the human system, we examined a panel of human cell lines for ALK expression and found that transcription is completely repressed in cell lines of entodermal origin (0/21). Furthermore, full length receptor expression is absent in cell lines of the hematopoietic system with the exception of t(2;5)-associated anaplastic large cell lymphomas lines (ALCL), which are known to express chimeric NPM-ALK mRNA. Cell lines established from solid tumors of ectodermal origin, including melanoma and breast carcinoma, exhibited widespread mRNA expression of the ALK receptor at a broad range (53/64), an association which was found to be strongest in cell lines derived from neuroblastoma (6/6), glioblastoma (8/8) as well as in cell lines established from Ewing sarcoma (4/4) and retinoblastomas (2/2). Because of the reported involvement of neutrophin tyrosine kinase receptors in autocrine differentiation in neuroblastomas, we analyzed cell lines positive for full length or chimeric ALK protein for the presence of phoshotyrosine residues within the intracellular region of ALK. While the constitutive activation of chimeric NPM-ALK molecules could be shown, no evidence was found for induced or constitutively activated ALK receptors in neuroblastoma, melanoma or breast carcinoma cell lines. Although the receptor could be shown to be consistently expressed with exclusive specificity in tissues developed from the ectoderm, our results do not support any involvement of ALK in the stimulation of tumorigenic cell growth or differentiation so far, indicating that ALK expression is a physiologic rather than a pathologic phenomenon.

Study about the inhibition of L-cysteine derivatives of nucleic acid bases in protein production.

Isopoly (S-carboxymethyl-L-cysteine) derivatives of nucleic acids bases were prepared as antisense compounds. In past study, we investigated the properties of these compounds in vitro, and revealed that these compounds in vivo regulated the cell death presumably due to the inhibition of protein production. In this study, western and northern blots were carried out in order to reveal the mechanism of this inhibition for N-methyl-D-aspartate receptor in neuroblastoma x glioma hybrid NG108-15 cell line. In addition, we investigated the resistance of these compounds against cell extract and the metabolism. In conclusion, we proved that these compounds inhibited the protein production by antisense mechanism.

Signal transduction pathways through TRK-A and TRK-B receptors in human neuroblastoma cells.

Little is known about the signal transduction pathways of TRK family receptors in neuroblastoma (NB) cells. In this study, an NB cell line, designated MP‐N‐TS, was established from an adrenal tumor taken from a 2‐year‐old boy. This cell line expressed both TRK‐A and TRK‐B receptors, which is rare in a single NB cell line. Therefore, the MP‐N‐TS cell line was used to determine whether the signal transduction through these constitutive receptors is functional. Three neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/ 5 (NT‐4/5), induced tyrosine phosphorylation of panTRK, and BDNF and NT‐4/5 induced tyrosine phosphorylation of TRK‐B. Tyrosine phosphorylation of panTRK and/or TRK‐B by the neurotro‐phins was inhibited in the presence of a tyrosine kinase inhibitor K252a. Tyrosine phosphorylation of Src homologous and collagen (She), extracellular signal‐regulated kinase (ERK)‐l and ERK‐2, and phospholipase C‐γl (PLC‐γl) was increased by the three neurotrophins and the increase was inhibited in the presence of K252a. Activation of Ras, detected as the GTP‐bound form of Ras, was induced by the three neurotrophins. The neurotrophins did not modulate the expressions of TRK‐A or TRK‐B mRNA, but they did induce the expression of c‐fos mRNA. Exogenous NGF induced weak neurite outgrowth, whereas exogenous BDNF and NT‐4/5 induced distinct neurite outgrowth. Exogenous BDNF and NT‐4/5 increased the number of viable cells, while NGF did not. Our results demonstrate that the signal transduction pathways through TRK‐A and TRK‐B in MP‐N‐TS cells are functional and similar, and the main downstream signaling pathways from the three neurotrophins are mitogen‐activated protein kinase (MAPK) cascades through She, activated Ras, ERK‐1 and ERK‐2, and the transduction pathway through PLC‐γl. Further, BDNF and NT‐4/5 increased cell viability. The MP‐N‐TS cell line should be useful for clarifying the TRK‐A and TRK‐B signaling pathways responsible for the different prognoses in patients with NB.

Decreased wild‐type full‐length Et‐A and ‐B receptors in neuroblastoma and Ewing sarcoma cells

Decreased wild‐type full‐length Et‐A and ‐B receptors in neuroblastoma and Ewing sarcoma cells

Decreased wild-type full-length Et-A and -B receptors in neuroblastoma and Ewing sarcoma cells

Endothelins and their receptors, Et-A and Et-B, play an essential role in differentiation and migration of neural crest cells. Expression of endothelin receptors has been examined in neuroblastoma and Ewing sarcoma cell lines. RNA was amplified for Et-A and Et-B by RT-PCR. Amplified products were cloned into the expression vector pLNCX, which was used to transfect CHO cells. Binding characteristics of transfected CHO cells were examined. Full-length Et-A mRNA was identified in all cell lines, in addition to a truncated Et-A product. CHO cells expressing full-length Et-A bound to endothelin, but cells expressing truncated Et-A did not. Full length Et-B mRNA was not detected, but two smaller molecular weight products were amplified. These are as yet uncharacterised. These results suggest that endothelins and their receptors may be important in the development and biology of neuroblastoma and Ewing sarcoma.

ANGIOGENESIS AND NEUROBLASTOMAS: INTERLEUKIN-8 AND INTERLEUKIN-8 RECEPTOR EXPRESSION IN HUMAN NEUROBLASTOMA

Studies have demonstrated that the pro-angiogenic cytokine interleukin-8 (IL-8) and the IL-8 receptors likely have a role in the growth and metastasis of various solid tumors. We hypothesized that in vivo neuroblastoma expresses IL-8 and the IL-8 receptors A and B, and that factors known to regulate IL-8 expression are present and active in the neuroblastoma microenvironment. To confirm the presence of IL-8/IL-8 receptors in neuroblastoma, immunohistochemical analysis for IL-8 and its receptors was performed on 10 archival specimens, including benign adrenal and well to poorly differentiated neuroblastoma samples. Immunohistochemical analysis was also performed for interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha. Cultured neuroblastoma cells SK-N-MC and SK-N-SH were stimulated with 10 ng./ml. IL-1beta or tumor necrosis factor-alpha and control media (15 each). Cell culture supernatants were analyzed with enzyme-linked immunosorbant assay for IL-8 levels at 24 and 48 hours. Minimal expression of IL-8 was noted in benign adrenal tissue but expression for IL-8 was present in all neuroblastoma specimens. Microvessel staining was present in 30% of the specimens. All tumor specimens expressed IL-8 receptor B, and both receptors were expressed in the tumor microvasculature. Immunohistochemical analysis confirmed the presence of IL-1beta and tumor necrosis factor in the neuroblastoma microenvironment. In vitro studies demonstrated that SK-N-MC and SK-N-SH cells express low levels of IL-8 under normal conditions and that IL-1beta and tumor necrosis factor-alpha significantly increased expression of IL-8 at 24 and 48 hours. Our results indicate that IL-8 and its receptors are expressed in neuroblastoma tumor specimens. In addition, the fact that IL-1beta and tumor necrosis factor-alpha are expressed in the neuroblastoma microenvironment combined with our in vitro results suggests that these cytokines may be involved in in vivo regulation of IL-8 in human neuroblastoma. Understanding the angiogenic factors and regulatory cascade promoting angiogensis in neuroblastoma may lead to the development of effective anti-angiogenic strategies.

ANGIOGENESIS AND NEUROBLASTOMAS: INTERLEUKIN-8 AND INTERLEUKIN-8 RECEPTOR EXPRESSION IN HUMAN NEUROBLASTOMA

Purpose: Studies have demonstrated that the pro-angiogenic cytokine interleukin-8 (IL-8) and the IL-8 receptors likely have a role in the growth and metastasis of various solid tumors. We hypothesized that in vivo neuroblastoma expresses IL-8 and the IL-8 receptors A and B, and that factors known to regulate IL-8 expression are present and active in the neuroblastoma microenvironment.Materials and Methods: To confirm the presence of IL-8/IL-8 receptors in neuroblastoma, immunohistochemical analysis for IL-8 and its receptors was performed on 10 archival specimens, including benign adrenal and well to poorly differentiated neuroblastoma samples. Immunohistochemical analysis was also performed for interleukin-1β (IL-1β) and tumor necrosis factor-α. Cultured neuroblastoma cells SK-N-MC and SK-N-SH were stimulated with 10 ng./ml. IL-1β or tumor necrosis factor-α and control media (15 each). Cell culture supernatants were analyzed with enzyme-linked immunosorbant assay for IL-8 levels at 24 and 48 hours.Result...

A DPDPE-induced enhancement of inward rectifier potassium current via opioid receptor in neuroblastoma×glioma NG108-15 cells

The effect of a delta-selective opioid agonist, DPDPE([D-Pen(2, 5)]-enkephalin), on the inward rectifier potassium current (I(KIR)) of NG108-15 cell was studied by whole cell voltage-clamp technique. It was found that microM DPDPE increased the amplitude and delayed the activation and inactivation of I(KIR) rapidly and reversibly. These effects could be reversed by naloxone, but were still obtained in pertussis toxin (PTX) preincubated cells or when using GDP-betas (guanosine 5'-o-[2-thio] diphoaphate) containing electrodes to block the G-protein coupled events. The above results suggest that DPDPE-induced change of I(KIR) is mediated by delta-opioid receptor but does not involve G-proteins.