Growth Of Human Neuroblastoma Research Articles

Royal jelly lipophilic fraction induces antiproliferative effects on SH-SY5Y human neuroblastoma cells.

Royal jelly (RJ) is one the most important bee product because it strongly influences the larval development in the hive, including the queen bee. In literature, RJ is known for its antioxidant, immunoregulatory, antifungal, antibiotical, erythropoietic, hypoglycemic, anticholesteremic, antithyroidic, anti-osteoporotic and estrogenic properties. However, it is surprising how rare the scientific evidence about RJ antineoplastic capacity are. That being said, we investigated, for the first time, the invitro bioactivity of six different RJs on the growth of three different mammalian cell lines: immortalized murine myoblasts (C2C12), human prostate cancer (PC3) and human neuroblastoma (SH-SY5Y). These studies were performed treating the cells with the only lipophilic, or hydrophilic, fraction of the RJs, a scientific approach never performed before. Moreover, chemical and protein profiles of all RJs were finely characterized, in qualitative and quantitative terms, by GC-MS and 1D-SDS-PAGE, respectively, in order to give a complete framework to the research. Despite the deep differences we found in the composition of each sample, unexpectedly, RJs showed comparable or very similar biological effects. In particular, our attention was captured by the extraordinary antiproliferative activity of the lipophilic extract of all RJs against SH-SY5Y cells, suggesting a potential medical application of this bee product to prevent the onset and slow down the growth of human neuroblastoma.

Efficacy of focal adhesion kinase inhibition in non-small cell lung cancer with oncogenically activated MAPK pathways.

Background:Focal adhesion kinase (FAK) is overexpressed in many types of tumours, including lung cancer. Y15, a small molecule which inhibits Y397 FAK autophosphorylation, decreases growth of human neuroblastoma, breast and pancreatic cancers. In this study, we investigated the in vitro and in vivo effects of Y15, and the underlying mechanism on non-small cell lung cancer cells.Methods:The cytotoxic effects of Y15 targeting FAK signalling were evaluated. Gene-knockdown experiments were performed to determine the anti-cancer mechanism. Xenografts with RAS or EGFR mutations were selected for in vivo Y15 treatment.Results:Y15 blocked autophosphorylation of FAK in a time- and dose-dependent manner. It caused dose-dependent decrease of lung cancer cell viability and clonogenicity. Y15 inhibited tumour growth of RAS-mutant (A549 with KRAS mutation and H1299 with NRAS mutation), as well as epidermal growth factor receptor (EGFR) mutant (H1650 and H1975) lung cancer xenografts. JNK activation is a mechanism underlying Y15-induced Bcl-2 and Mcl-1 downregulation. Moreover, knockdown of Bcl-2 or Bcl-xL potentiated the effects of Y15. The combination of various inhibitors of the Bcl-2 family of proteins with FAK inhibitors demonstrated synergy in multiple lung cancer cell lines in vitro.Conclusions:FAK inhibition demonstrated efficacy both in vitro and in vivo in lung cancers with either oncogenic RAS or EGFR mutations. In addition, FAK inhibition in combination with inhibitors of Bcl-2 family of anti-apoptotic proteins has synergistic activity in these MAPK-activated non-small cell lung cancer cell line models.

Targeting high-risk pediatric solid tumors with CAR T cells directed against ALK (anaplastic lymphoma kinase, CD246)

Neuroblastoma is the most common non-CNS tumor in children. Poor prognosis for patients presenting with high-risk disease underlines the need for novel therapeutic strategies. Chimeric antigen receptor T cell (CART) therapies combine the epitope specificity of a monoclonal antibody with the cytotoxicity of an activated T cell, and have demonstrated clinical efficacy in pediatric leukemia. To expand this approach to neuroblastoma, we focused on anaplastic lymphoma kinase (ALK), which can exist in native, over-expressed, or mutated forms and may help drive disease progression. Single chain variable fragments (scFvs) derived from four different monoclonal antibodies against ALK were used to construct second-generation CAR vectors featuring either CD28/CD3-zeta or CD137/CD3-zeta signaling domains. Some CAR constructs also included an IgG1 CH2CH3 spacer domain between the scFv and transmembrane domain. Retroviral vectors expressing ALK-specific CARs were generated and used to transduce human peripheral blood mononuclear cells. Transduction efficiencies ranged from 8% to 99%, depending on specific characteristics of each CAR construct. The resulting CART lysed ALK-positive neuroblastoma and Ewing's sarcoma cell lines and had minimal activity against an ALK-negative control. The ability to lyse tumor cells varied across scFvs, and short CART (no Ig spacer) showed enhanced lysis compared to long CARs (with Ig spacer). Short CART also produced Th1 cytokines (including IFN-gamma, IL-2, and TNF-alpha) when co-incubated with ALK-positive tumor lines; however, long CARs did not. Additionally, short anti-ALK CART significantly delayed the growth of human neuroblastoma in an NSG xenograft model when tumor-bearing mice were preconditioned with cyclophosphamide. Ongoing studies aim to: 1) further assess anti-ALK CART therapy in vivo in combination with modulation of suppressive murine myeloid populations; 2) explore mechanisms of ALK-positive neuroblastoma resistance to CART-mediated eradication; 3) characterize ALK protein expression in murine central nervous system tissues to better understand possibility of on-target, off-tumor toxicities with this therapy. Ultimately, anti-ALK CART could provide a novel therapy for pediatric solid tumors expressing cell-surface ALK.

Tryptanthrin induces growth inhibition and neuronal differentiation in the human neuroblastoma LA-N-1 cells

Neuroblastoma is one of the most common extracranial solid cancers found in young children. The prognosis of neuroblastoma patients in advanced stages having N-myc amplification remains poor despite intensive multimodal therapy. Agents that trigger neuroblastoma cells to undergo cellular differentiation and thereby stop proliferation have attracted considerable interest as an alternative therapy. Tryptanthrin (12-dihydro-6,12-dioxoindolo-(2,1-b)-quinazoline) is a weakly basic alkaloid isolated from the dried roots of medicinal indigo plants known as Banlangen. It has been shown to possess various biological activities, such as anti-microbial, anti-inflammatory and anti-tumor activities. However, its effects and mechanism(s) of action on human neuroblastoma cells remain poorly understood. Therefore, the objective of this study is to investigate the effects of tryptanthrin on the growth and differentiation of human neuroblastoma LA-N-1 cells with N-myc amplification. Our results show that tryptanthrin inhibited the growth of the human neuroblastoma cells in a dose- and time-dependent manner. Mechanistic studies indicated that tryptanthrin induced cell cycle arrest of the human neuroblastoma LA-N-1 cells at the G0/G1 phase. Tryptanthrin also induced neuronal differentiation of LA-N-1 cells, as assessed by morphological criteria, enhancement of acetylcholine esterase activity and up-regulation of various differentiation markers. Moreover, tryptanthrin treatment led to the significant reduction of N-myc expression in LA-N-1 cells while siRNA directed against N-myc induced morphological differentiation of LA-N-1 cells. These results, when taken together, suggest that tryptanthrin suppressed the growth and induced neuronal differentiation in the human neuroblastoma LA-N-1 cells and might be exploited as a potential therapeutic candidate for the treatment of high-risk neuroblastomas with N-myc-amplification.

Chd5 Requires PHD-Mediated Histone 3 Binding for Tumor Suppression

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

Tryptamine-Induced Tryptophanyl-tRNAtrp Deficiency in Neurodifferentiation and Neurodegeneration Interplay: Progenitor Activation with Neurite Growth Terminated in Alzheimer's Disease Neuronal Vesicularization and Fragmentation

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes tryptophanyl-tRNAtrp formation. At concentrations exceeding tryptophan, tryptamine inhibits TrpRS. This leads in tryptophanyl-tRNA deficiency and synthesis of aberrant proteins. Tryptamine presents in food and crosses blood-brain barrier. The purpose of this study is to test the hypothesis that tryptamine-induced changes in cell and animal models correlate with Alzheimer's disease (AD) manifestations. Tryptamine prevented growth of human neuroblastoma. Epithelioids recovered growth in tryptamine-free medium, while neuroblasts died. Tryptamine induced epithelioid differentiation forming synaptic vesicles, neuritic contacts, and TrpRS+ axons in stable sublines. A fraction of epithelioids was adhered to satellite cells via trypsin-resistant interdigitating junctions. Tryptamine stimulated satellite division and differentiation into neurons, transitional cell variants and neuroblasts able to repopulate. Both tryptamine-inhibited and hypoxia-downregulated TrpRS translocates into cytoplasmic extensions. TrpRS is secreted into extracellular space as a free protein or within vesicles extended from cytoplasm and then pinched-off from plasma membrane of tryptamine-treated cells. Extracellular vesicles fuse in congophilic TrpRS+ plaques in tryptamine-treated culture and AD brain. TrpRS prominent immunoreactivity is associated with plasma and vesicle membranes of satellites and AD brain degenerated neurons. Tryptamine-modified mouse brain expresses amyloid and abnormal filaments in extracellular and neuronal plasma membrane vesicles. Radiolabeled tryptamine, tryptophan and serotonin uptake was 10-fold lower in tryptamine-resistant compared to tryptamine-sensitive cells. In both variants, tryptamine uptake exceeded tryptophan uptake within 2-h assuring TrpRS inhibition. Here, tryptophanyl-tRNAtrp deficiency implicates in both neurite growth and termination/collapse. Neurite growth termination prompts TrpRS+ vesicularization. TrpRS+ vesicles contribute in neuronal fragmentation and fibrillar-vesicular congophilic plaques in AD brain.

Resveratrol Inhibits Tumor Growth of Human Neuroblastoma and Mediates Apoptosis by Directly Targeting Mitochondria

Neuroblastoma is an aggressive childhood disease of the sympathetic nervous system. Treatments are often ineffective and have serious side effects. Because resveratrol, a natural plant product, has been reported to have limited toxicity at chemotherapeutic levels, we investigated its efficacy in the treatment of neuroblastoma as well as its underlying mechanism of action. Resveratrol was tested in mouse xenograft models of human neuroblastoma and in vitro using human cell lines. Resveratrol inhibited the outgrowth of tumors by as much as 80%. The bioavailability of the drug in serum was in the low micromolar range (2-10 micromol/L) and no accumulation was observed in tumor tissue. When resveratrol levels were increased by peritumor injection, rapid tumor regression occurred. Resveratrol decreased tumor cell viability in vitro by 75% to 90%, resulting from an inhibition of cell proliferation and an induction of apoptosis. Loss of mitochondrial membrane potential was an early response to resveratrol. In addition, resveratrol treatment of isolated mitochondria also led to depolarization, suggesting that the drug may target mitochondria directly. Following depolarization, resveratrol caused the release of cytochrome c and Smac/Diablo from the mitochondria and subsequently the activation of caspase-9 (4- to 8-fold) and caspase-3 (4- to 6-fold). These studies indicate that, despite low bioavailability, resveratrol is effective at inhibiting tumor growth. Elevated levels of resveratrol enhance its antitumor potency leading to tumor regression, associated with widespread tumor cell death, the underlying mechanism of which involves the direct activation of the mitochondrial intrinsic apoptotic pathway.

Combined Therapeutic Effects of Vinblastine and Rapamycin on Human Neuroblastoma Growth, Apoptosis, and Angiogenesis

Vinblastine and rapamycin displayed synergistic inhibition of human neuroblastoma-related angiogenesis. Here, we studied the antitumor activity of vinblastine and rapamycin against human neuroblastoma. Cell proliferation, cell cycle progression, and apoptosis were evaluated by measuring (3)H-thymidine incorporation, bromodeoxyuridine uptake, and phosphatidylserine exposure, respectively. The in vivo sensitivity of neuroblastoma cells to vinblastine and rapamycin was determined in orthotopic neuroblastoma-engrafted mice. Angiogenesis was assessed by the chick embryo chorioallantoic membrane assay. Each compound alone was able to induce a dose-dependent significant inhibition of cell proliferation, with a dramatically enhanced antiproliferative effect for the drugs used in combination. A marked G(2)-M cell cycle arrest with a nearly complete depletion of S phase was associated. The combined treatment triggered an increased apoptosis compared with either drug tested alone. A significant inhibition of tumor growth and microvessel area was obtained in neuroblastoma-bearing mice when treated with vinblastine or rapamycin alone, and a more dramatic effect with the combined treatment, compared with control mice. The therapeutic effectiveness, expressed as increased life span, was statistically improved by the combined therapy, compared with mice treated with either drug tested separately. Histologic evaluation of primary tumors showed that the combined treatment inhibited proliferation and angiogenesis and induced apoptosis. Combined treatment of neuroblastoma cells and neuroblastoma-bearing mice with vinblastine and rapamycin induced the down-modulation of both vascular endothelial growth factor production and vascular endothelial growth factor receptor 2 expression. In the chorioallantoic membrane assay, angiogenesis induced by human neuroblastoma biopsy specimens was significantly inhibited by vinblastine and rapamycin. These results may be relevant to design new therapeutic strategies against neuroblastoma.

1 α-Hydroxyvitamin D2 inhibits growth of human neuroblastoma

Neuroblastoma is the most common extracranial solid tumor in childhood. The poor outcomes of patients with high-risk neuroblastoma have encouraged the search for new therapies. In the current study, the effect of the vitamin D analog 1alpha-hydroxyvitamin D2 (1alpha-OH-D2, doxercalciferol) was assessed in a mouse xenograft model of human neuroblastoma. Vitamin D receptor (VDR) expression levels in seven neuroblastoma cell lines were compared using real-time PCR. SK-N-AS cells, which express relatively high levels of VDR, were injected into the flanks of 60 mice. The mice were treated daily via oral gavage for 5 weeks with vehicle (control), 0.15 microg, or 0.3 microg of 1alpha-OH-D2. The animals were then euthanized, and tumors, sera, and kidneys were collected and analyzed. End tumor volumes were significantly smaller in both the 0.15 microg group (712.07 mm3, P = 0.0121) and 0.3 microg group (772.97 mm3, P = 0.0209) when compared to controls (1,681.75 mm3). In terms of toxicity, serum calcium levels were increased but mortality was minimal in both treatment groups. These results were similar to those previously described in the transgenic (LHbeta-Tag) and human xenograft (Y-79) models of retinoblastoma, a related tumor. In vitro cell viability studies of SK-N-AS and NGP cells, which represent two major human neuroblastoma subtypes that differ in their genetic abnormalities as well as their VDR expression levels, show that both are sensitive to calcitriol, the active metabolite of vitamin D3. In conclusion, the present study shows that 1alpha-OH-D2 can inhibit human neuroblastoma growth in vivo with relatively low toxicity. The safety of 1alpha-OH-D2 has been extensively studied; the drug is FDA-approved for the treatment of adult kidney patients, and Phase I/II trials have been conducted in adult oncology patients. There should not be major obstacles to starting Phase I and II clinical trials with this drug in pediatric patients with high-risk neuroblastoma.

488 POSTER A cell-permeable dominant-negative Survivin protein as a tool to understand how Survivin maintains tumour cell survival

All-transretinoic acid (RA) reduces human neuroblastoma growth by inducing either differentiation or apoptosis. The apoptotic program in these cells is regulated by RA and is paralleled by the transcriptional induction of “tissue” transglutaminase (tTG). tTG is a protein cross-linking enzyme, which specifically accumulates in cells undergoing apoptosis in variousin vivoandin vitrosystems. In neuroblastoma cells, tTG is detected exclusively in the cells expressing the S-type phenotype and showing an increased apoptosis. The present study was undertaken to identify the retinoid receptors which are involved in the regulation of tTG and apoptosis as well as in thein vitroneuronal differentiation of the human SK-N-BE(2) neuroblastoma cell line. We have previously characterized the retinoid acid receptors expressed in this cell line. In the present study, by using synthetic retinoids selectively activating RAR/RXR isoforms, we have identified the RAR/RXR receptors involved in the induction of either apoptosis or differentiation. We have also studied the effect of the selective RA analogs on tTG activity. We observed that while RARα- and RARγ-selective retinoids alone were able to induce tTG activity, only the combined stimulation of both RARα and RARγ induced apoptosis. Conversely, several combinations of RAR/RXR closely mimicked the differentiation effects observed with all-transretinoic acid. These results indicate that, at variance with differentiation, the induction of apoptosis in human SK-N-BE(2) neuroblastoma cells is under the specific control of RARα and RARγ. These data seem relevant for the reported ability of RARγ to suppress the clinically malignant tumor phenotype in patients.

Methionine Aminopeptidase 2 Inhibition Is an Effective Treatment Strategy for Neuroblastoma in Preclinical Models

Tumor vascularity is correlated with an aggressive disease phenotype in neuroblastoma, suggesting that angiogenesis inhibitors may be a useful addition to current therapeutic strategies. We previously showed that the antiangiogenic compound TNP-470, an irreversible methionine aminopeptidase 2 (MetAP2) inhibitor, suppressed local and disseminated human neuroblastoma growth rates in murine models but had significant associated toxicity at the effective dose. We have recently shown that a novel, reversible MetAP2 inhibitor, A-357300, significantly inhibits CHP-134-derived neuroblastoma s.c. xenograft growth rate with a treatment-to-control (T/C) ratio at day 24 of 0.19 (P < 0.001) without toxicity. We now show that the combination of A-357300 with cyclophosphamide at the maximal tolerated dose sustained tumor regression with a T/C at day 48 of 0.16 (P < 0.001) in the CHP-134 xenograft model. A-357300 also significantly inhibited establishment and growth rate of hematogenous metastatic deposits following tail vein inoculation of CHP-134 cells and increased overall survival (P = 0.021). Lastly, A-357300 caused regression of established tumors in a genetically engineered murine model with progression-free survival in five of eight mice (P < 0.0001). There was no evidence of toxicity. These data show that MetAP2 may be an important molecular target for high-risk human neuroblastomas. We speculate that the growth inhibition may be through both tumor cell intrinsic and extrinsic (antiangiogenic) mechanisms. The potential for a wide therapeutic index may allow for treatment strategies that integrate MetAP2 inhibition with conventional cytotoxic compounds.

Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells

Phellinus linteus is a well-known Oriental medicinal fungus that has various biological activities, including immunomodulatory and anti-tumor activities, the mechanisms of which are poorly understood. In the present study, we investigated the effects of mycelium extracts of P. linteus (MEPL) on the growth of human neuroblastoma SK-N-MC cells. Upon treatment with MEPL, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis, including condensation of chromatin and an increase in the sub-G1 population. The anti-proliferative and apoptotic effects of MEPL were associated with a marked induction of the Bax and cyclin-dependent kinase inhibitor p21. Western blotting and in vitro caspase-3 activity assay demonstrated that the processing/activation of caspases accompanies the generation of MEPL-mediating apoptotic cell death. In addition, the proteolytic cleavage of specific target proteins such as poly(ADP-ribose) polymerase and beta-catenin were observed. Taken together, the present results suggest that apoptotic signals evoked by MEPL in human neuroblastoma SK-N-MC cells may converge caspase-3 activation through an up-regulation of Bax rather than a down-regulation of Bcl-2.

Effect of CEP-751 (KT-6587) on neuroblastoma xenografts expressing TrkB.

The compound CEP-751 (KT-6587), a potent and selective inhibitor of the Trk family of tyrosine kinases, has been shown to inhibit the growth of human neuroblastoma (NB) xenografts in nude mice [1]. To address its mechanism of action, we studied SY5Y, a human NB cell line with no detectable Trk expression, and two subclones transfected with TrkB. The transfected clones, SY5Y (G8) and SY5Y (G12), expressed moderate and high levels, respectively, of TrkB mRNA and protein. These TrkB-expressing subclones and the parental line were then grown as xenografts in nude mice, and CEP-751 was used to inhibit TrkB tyrosine kinase activity in these xenografts. Animals were treated twice a day with CEP-751 (21 mg/kg), or with the carrier vehicle as a control. TrkB expression in the resultant tumors was examined by quantitative RT-PCR. The effect of CEP-751 on TrkB activation by BDNF was examined in G12 cells in culture by immunoprecipitation with antipan Trk antiserum, followed by Western blot analysis using antiphosphotyrosine antibodies. To determine if CEP-751 was causing apoptosis, the TUNEL assay was used. CEP-751 had little effect on the growth of SY5Y tumors, but did slow the growth rate of the C8 and G12 tumors. The daily growth rate of the treated tumors was 0.16, 0.13, and 0.10 cm3, respectively, for the SY5Y, G8, and G12 tumors. RT PCR analysis confirmed the expression of TrkB in G8 and G12, but not in SY5Y tumors. Activation of TrkB by BDNF in G12 cells was inhibited by CEP-751 in a dose dependent fashion. The treated tumors showed marked evidence of apoptosis. These data suggest that the effect of CEP-751 is due, at least in part, to its inhibition of TrkB kinase, and that CEP-751 may become a useful therapeutic tool for the treatment of aggressive neuroblastomas, which often express TrkB.

Antiangiogenic therapy inhibits human neuroblastoma growth.

Angiogenesis plays a crucial role in the progression and metastasis of malignant solid tumors. In many types of cancer, including neuroblastoma, high tumor vascularity correlates with poor outcome. Recently, a number of angiogenic inhibitors have been identified that had antitumor activity in preclinical studies. To investigate the effect of the antiangiogenic agent TNP-470 on neuroblastoma tumor growth, we treated nude mice with TNP-470 after they were inoculated subcutaneously with 5 x 10(6) cells from the MYCN-amplified, human neuroblastoma cell line NBL-W-N. TNP-470 did not significantly affect tumor growth when it was administered to mice with large tumors (>600 mm3). However, when TNP-470 was administered in the setting of microscopic disease 12 hr following tumor cell inoculation, treated mice had a significantly improved tumor-free survival at 12 weeks (P < 0.001), and overall survival at 45 weeks (P < 0.001), compared to control animals. Our studies suggest that TNP-470 treatment may be most effective if it is administered in the setting of microscopic disease. We speculate that TNP-470 may inhibit neuroblastoma growth in children if treatment is initiated following intensive multimodality therapy, when residual disease is minimal.

Histone deacetylase inhibitors and retinoic acids inhibit growth of human neuroblastoma in vitro.

Neuroblastoma is a common childhood cancer with a poor overall prognosis. Retinoic acids (RAs) have been studied as a potential therapy, showing promise in recurrent disease. The histone deacetylase inhibitor (HDACI) M-carboxycinnamic acid bishydroxamide (CBHA) is another potential therapy, which we recently described. Combinations of RAs and HDACIs currently under investigation display synergy in certain neoplasms. In this study, we evaluate the effect of combinations of RAs and HDACIs on human neuroblastoma cells. Established cell lines were cultured in increasing concentrations of HDACIs, RAs, and combinations thereof. Following exposure, viable cell number was quantified by trypan blue dye exclusion on a hemacytometer. Cell cycle analysis was performed by propidium iodide staining and FACS. All assayed HDACIs and RAs decreased viable cell number. Lower concentrations of each agent were effective when the two were combined. The primary reason for decreased cell number appears to be apoptosis following HDACI exposure and G1 arrest following RA exposure. Both effects are seen with cotreatment. Caspase inhibition abrogates the apoptotic response. CBHA causes apoptosis of human neuroblastoma in vitro, an effect that can add to the effects of RA. HDACIs and RAs inhibit neuroblastoma in significantly lower concentrations when used together than when used individually. Combination therapy may improve the ultimate efficacy while reducing the side effects of these agents in clinical use.

Inhibition of tumor growth in a human neuroblastoma xenograft model with TNP-470.

Background and Procedure High-risk neuroblastoma disease features are correlated with tumor vascularity, suggesting that angiogenesis inhibitors may be a useful addition to current therapeutic strategies. We therefore examined the efficacy of TNP-470 (TAP Pharmaceuticals, Deerfield, IL) in human neuroblastoma xenograft models. Tumor growth rate was markedly inhibited in mice receiving TNP-470 administered alone with a treatment to control ratio (T/C) at day 21 = 0.4 (P <.001). TNP-470 also significantly inhibited tumorigenicity when administered shortly after xenograft inoculation (T/C at day 30 = 0.1, P <.001) and when administered following cyclophosphamide (T/C at day 35 = 0.1, P <.001). These data show that TNP-470 is a potent inhibitor of human neuroblastoma growth both alone and when given with conventional chemotherapy, suggesting that it may be a useful adjunctive therapy for high-risk neuroblastoma patients.

Human neuroblastoma cell growth in tissue culture is regulated by opioid growth factor.

Human neuroblastoma is one of the most common solid tumors in infants and children and represents about 10% of all childhood cancers. Nearly 70% of the patients present with disseminated disease and the long-term prognosis remains poor for this group despite advances in diagnosis and therapy. In this study, we discovered that an endogenous opioid peptide, [Met5]-enkephalin, inhibited the growth of human neuroblastoma SK-N-SH in vitro; in view of this pentapeptide's action it has been termed opioid growth factor (OGF). OGF was found to be constitutively expressed and tonically capable of suppressing cell replication, and its effects were opioid receptor mediated. Growth inhibition was dose-related, reversible, not cytotoxic, and independent of serum. Immunocytochemical studies detected both OGF and its related receptor, zeta, in the cytoplasm of log-phase cells. Pharmacological binding assays revealed specific and saturable binding with a one-site model of kinetics; this high-affinity opioid receptor was identified as zeta. These data suggest that a native opioid peptide, OGF, interacts with a novel opioid receptor, zeta, to arrest the growth of human neuroblastoma.

Retinoic Acid Receptors α and γ Mediate the Induction of “Tissue” Transglutaminase Activity and Apoptosis in Human Neuroblastoma Cells

All-transretinoic acid (RA) reduces human neuroblastoma growth by inducing either differentiation or apoptosis. The apoptotic program in these cells is regulated by RA and is paralleled by the transcriptional induction of “tissue” transglutaminase (tTG). tTG is a protein cross-linking enzyme, which specifically accumulates in cells undergoing apoptosis in variousin vivoandin vitrosystems. In neuroblastoma cells, tTG is detected exclusively in the cells expressing the S-type phenotype and showing an increased apoptosis. The present study was undertaken to identify the retinoid receptors which are involved in the regulation of tTG and apoptosis as well as in thein vitroneuronal differentiation of the human SK-N-BE(2) neuroblastoma cell line. We have previously characterized the retinoid acid receptors expressed in this cell line. In the present study, by using synthetic retinoids selectively activating RAR/RXR isoforms, we have identified the RAR/RXR receptors involved in the induction of either apoptosis or differentiation. We have also studied the effect of the selective RA analogs on tTG activity. We observed that while RARα- and RARγ-selective retinoids alone were able to induce tTG activity, only the combined stimulation of both RARα and RARγ induced apoptosis. Conversely, several combinations of RAR/RXR closely mimicked the differentiation effects observed with all-transretinoic acid. These results indicate that, at variance with differentiation, the induction of apoptosis in human SK-N-BE(2) neuroblastoma cells is under the specific control of RARα and RARγ. These data seem relevant for the reported ability of RARγ to suppress the clinically malignant tumor phenotype in patients.

A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice.

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.

Human neuroblastoma growth inhibitory factor (h-NGIF), derived from human astrocytoma conditioned meduim, has neurotrophic properties

Investigations on the general characteristics of human astrocytoma cell line NAC-1 revealed neuroblastoma growth inhibitory activity in conditioned medium. Neuroblastoma growth inhibitory factor (NGIF) was partially purified by Econo Q, Econo CM, and Superose 12 column chromatography. The protein is weakly basic with an estimated M r of 120,000, possibly having an M r 60,000 dimeric structure. NGIF inhibits the growth of neurobalstoma cell lines but has no effect on morphology nor does it produce any change in the growth of human glioblastoma cell lines. Interstingly, NGIF appears to promote survival and neurite outgrowth of embryonal rat cortical neurons. These neurotrophic properties suggest a role for NGIF in the development of the nervous system.