Proliferation Of Human Neuroblastoma Cells Research Articles

Up-regulation of lncRNA WT1-AS ameliorates Aβ-stimulated neuronal injury through modulation of miR-186-5p/CCND2 axis in Alzheimer's disease.

As a common neurodegenerative disorder, Alzheimer's disease (AD) seriously threatens human life. Long non-coding RNAs (lncRNAs) exhibit essential functions in AD development. Nevertheless, the detailed effects and possible mechanisms of lncRNA Wilms tumor 1 Antisense RNA (WT1-AS) in AD are largely unknown. In our studies, a total of 30 serum samples from AD patients were collected, and WT1-AS expressions were detected through qRT-PCR analysis. Additionally, an in vitro AD model was constructed by treating Aβ1-42 in human neuroblastoma cells. Functional assays were implemented to assess the impacts of WT1-AS on Aβ1-42-stimulated human neuroblastoma cell proliferation together with apoptosis. Moreover, relationship of WT1-AS, microRNA (miR)-186-5p as well as cyclin D2 (CCND2) could be predicted through bioinformatics tools as well as proved via dual-luciferase reporter experiments. Our results showed that WT1-AS together with CCND2 were low-expressed, while miR-186-5p presented high expression in AD serum samples together with Aβ1-42-stimulated human neuroblastoma cells. WT1-AS over-expression or miR-186-5p depletion notably promoted the proliferation, reduced the apoptosis, and decreased the p-Tau protein expressions of human neuroblastoma cells induced with Aβ1-42. Moreover, miR-186-5p combined with WT1-AS, and CCND2 was modulated by miR-186-5p. Furthermore, CCND2 elevation partially offsets the impacts of miR-186-5p elevation on Aβ1-42-stimulated cell proliferation as well as apoptosis mediated with WT1-AS up-regulation. Our results indicated that up-regulation of lncRNA WT1-AS ameliorated Aβ-stimulated neuronal damage through modulating miR-186-5p/CCND2 axis, offering a novel direction for AD therapy.

Monensin, an Antibiotic Isolated from Streptomyces Cinnamonensis, Regulates Human Neuroblastoma Cell Proliferation via the PI3K/AKT Signaling Pathway and Acts Synergistically with Rapamycin

Neuroblastoma is the most common extracranial childhood tumor and accounts for approximately 15% of pediatric cancer-related deaths. Further studies are needed to identify potential therapeutic targets for neuroblastoma. Monensin is an ionophore antibiotic obtained from Streptomyces cinnamonensis with known antibacterial and antiparasitic effects. No study has reported the effects of monensin on SH-SY5Y neuroblastoma cells by targeting the PI3K/AKT signaling pathway. The aim of this study was to investigate the antiproliferative effects of monensin alone and in combination with rapamycin in human SH-SY5Y neuroblastoma cells mediated by the PI3K/AKT signaling pathway. The effects of single and combination applications of monensin and rapamycin on SH-SY5Y cell proliferation were investigated by XTT, and their effects on the PI3K/AKT signaling pathway by RT-PCR, immunohistochemistry, immunofluorescence, and Western blotting. The combined effects of monensin and rapamycin on SH-SY5Y proliferation were most potent at 72 h (combination index < 1). The combination of monensin and rapamycin caused a significant decrease in the expression of P21RAS, AKT, and MAPK1 genes. Single and combined administrations of monensin and rapamycin caused a significant decrease in PI3K/AKT expression. Our results showed for the first time that monensin exerts an antiproliferative effect by targeting the PI3K/AKT signaling pathway in neuroblastoma cells. It is suggested that monensin and its combination with rapamycin may be an effective therapeutic candidate for treating neuroblastoma.

Luteoloside Induces G0/G1 Phase Arrest of Neuroblastoma Cells by Targeting p38 MAPK.

Luteoloside has shown anti-inflammatory, antiviral, and antitumor properties. However, the effect and mechanism of luteoloside on neuroblastoma cells remain unknown. The proliferation of human neuroblastoma cells (SH-SY5Y and SK-N-AS) treated with different concentrations of luteoloside (0, 12.5, 25, and 50 μM) was detected by the MTT assay and colony formation assay. Cell apoptosis and cell cycle were examined by Hoechst staining and flow cytometry. A subcutaneous tumorigenesis model was established in nude mice to evaluate the effect of luteoloside on tumor growth in vivo. Bioinformatics, molecular docking techniques, and cellular thermal shift assays were utilized to predict the potential targets of luteoloside in neuroblastoma. The p38 MAPK inhibitor SB203580 was used to confirm the role of p38 MAPK. Luteoloside inhibited the proliferation of neuroblastoma cells in vitro and in vivo. Luteoloside slightly induced cellular G0/G1 phase arrest and reduced the expression levels of G0/G1 phase-related genes and the proteins cyclin D1, CDK4, and C-myc, which are downregulated by p38 MAPK pathways. Meanwhile, p38 was identified as the target of luteoloside, and inhibition of p38 MAPK reversed the inhibitory effect of luteoloside on neuroblastoma cells. Luteoloside is a potential anticancer drug for treating neuroblastoma by activating p38 MAPK.

Field exposure to 50 Hz significantly affects wild-type and unfolded p53 expression in NB69 neuroblastoma cells.

Previous studies have shown that intermittent exposure to a 50 Hz, 100 µT sinusoidal magnetic field (MF) promotes proliferation of human neuroblastoma cells, NB69. This effect is mediated by activation of the epidermal growth factor receptor through a free radical-dependent activation of the p38 pathway. The present study investigated the possibility that the oxidative stress-sensitive protein p53 is a potential target of the MF, and that field exposure can affect the protein expression. To that end, NB69 cells were exposed to short intervals of 30 to 120 min to the aforementioned MF parameters. Two specific anti-p53 antibodies that allow discrimination between the wild and unfolded forms of p53 were used to study the expression and cellular distribution of both isoforms of the protein. The expression of the antiapoptotic protein Bcl-2, whose regulation is mediated by p53, was also analyzed. The obtained results revealed that MF exposure induced increases in p53 gene expression and in protein expression of the wild-type form of p53. Field exposure also caused overexpression of the unfolded form of p53, together with changes in the nuclear/cytoplasmic distribution of both forms of the protein. The expression of protein Bcl-2 was also significantly increased in response to the MF. As a whole, these results indicated that the MF is capable of interacting with the function, distribution and conformation of protein p53. Such interactions could be involved in previously reported MF effects on NB69 proliferation promotion.

Fabrication and Characterization of Nanofibrous Poly (L-Lactic Acid)/Chitosan-Based Scaffold by Liquid-Liquid Phase Separation Technique for Nerve Tissue Engineering.

Fabrication method is one of the essential factors which directly affect on the properties of scaffold. Several techniques have been well established to fabricate nanofibrous scaffolds such as electrospinning. However, preparing a three-dimensional (3-D) interconnected macro-pore scaffold essential for transporting the cell metabolites and nutrients is difficult using the electrospinning method. The main aim of this study was developing a highly porous scaffold by poly (L-lactic acid) (PLLA)/chitosan blend using liquid-liquid phase separation (LLPS) technique, a fast and cost-benefit method, in order to use in nerve tissue engineering. In addition, the effect of different polymeric concentrations on morphology, mechanical properties, hydrophilicity, in vitro degradation rate and pH alteration of the scaffolds were evaluated. Moreover, cell attachment, cell viability and cell proliferation of scaffolds as candidates for nerve tissue engineering was investigated. PLLA/chitosan blend not only had desirable structural properties, porosity, hydrophilicity, mechanical properties, degradation rate and pH alteration but also provided a favorable environment for attachment, viability, and proliferation of human neuroblastoma cells, exhibiting significant potential for nerve tissue engineering applications. However, the polymeric concentration in blend fabrication had influence on both characteristics and cell responses. It concluded that PLLA/chitosan nanofibrous 3-D scaffold fabricated by LLPS method as a suitable candidate for nerve tissue engineering.

Ubiquitination of p21 by E3 Ligase TRIM21 Promotes the Proliferation of Human Neuroblastoma Cells.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, which shows great clinical and biomolecule heterogeneity. Currently, surgery is still the main method of neuroblastoma treatment and specific therapeutic drugs are lacking, so useful targets are urgently needed. TRIM21 is a RING-type E3 ligase that its overexpression promotes the progression of human glioma, while whose effects on neuroblastoma have not been illustrated. Firstly, the shRNAs targeting TRIM21 were designed and found that the ablation of TRIM21 inhibits the proliferation of human neuroblastoma cells. Then the molecular mechanism study indicated that TRIM21 interacts with, and mediates p21 degradation by ubiquitination modification. Further study demonstrates that TRIM21 regulates the proliferation of neuroblastoma cells in a p21-dependent manner. These results suggest that TRIM21 might be a potential therapeutic target for neuroblastoma.

Inhibition of Human Neuroblastoma Cell Proliferation by N-acetyl-L-cysteine as a Result of Increased Sulfane Sulfur Level.

In various cancer cells, the level of sulfane sulfur-containing compounds is decreased compared to normal cells. In the present study the effect of N-acetyl-L-cysteine (NAC), which acts as a precursor of H2S synthesis, on the human neuroblastoma SH-SY5Y cell proliferation, the activity of 3-mercaptopyruvate sulfurtransferase (MPST), and the level of sulfane sulfur were investigated. SH-SY5Y cells were treated with NAC, while untreated cells were used as the control. The toxicity of NAC on the cells was studied by the LDH cytotoxicity assay; cell proliferation was examined by the MTT method. MPST activity and sulfane sulfur level were also analyzed in the NAC-treated cells. The addition of NAC to the medium, in non-cytotoxic concentrations, resulted in inhibition of the SH-SY5Y cell proliferation after 48 h of culture. The MPST activity and the level of sulfane sulfur-containing compounds were also elevated under the same culture conditions. The antiproliferative activity of NAC in the SH-SY5Y cells was associated with an increase in the MPST activity and consequently with an increase in the intracellular level of sulfane sulfur in these cells.

Identification of linc-NeD125, a novel long non coding RNA that hosts miR-125b-1 and negatively controls proliferation of human neuroblastoma cells

The human genome contains some thousands of long non coding RNAs (lncRNAs). Many of these transcripts are presently considered crucial regulators of gene expression and functionally implicated in developmental processes in Eukaryotes. Notably, despite a huge number of lncRNAs are expressed in the Central Nervous System (CNS), only a few of them have been characterized in terms of molecular structure, gene expression regulation and function. In the present study, we identify linc-NeD125 as a novel cytoplasmic, neuronal-induced long intergenic non coding RNA (lincRNA). Linc-NeD125 represents the host gene for miR-125b-1, a microRNA with an established role as negative regulator of human neuroblastoma cell proliferation. Here, we demonstrate that these two overlapping non coding RNAs are coordinately induced during in vitro neuronal differentiation, and that their expression is regulated by different mechanisms. While the production of miR-125b-1 relies on transcriptional regulation, linc-NeD125 is controlled at the post-transcriptional level, through modulation of its stability. We also demonstrate that linc-NeD125 functions independently of the hosted microRNA, by reducing cell proliferation and activating the antiapoptotic factor BCL-2.

Binding of Tissue-type Plasminogen Activator to the Glucose-regulated Protein 78 (GRP78) Modulates Plasminogen Activation and Promotes Human Neuroblastoma Cell Proliferation in Vitro

The glucose-regulated protein 78 (GRP78) is a plasminogen (Pg) receptor on the cell surface. In this study, we demonstrate that GRP78 also binds the tissue-type plasminogen activator (t-PA), which results in a decrease in K(m) and an increase in the V(max) for both its amidolytic activity and activation of its substrate, Pg. This results in accelerated Pg activation when GRP78, t-PA, and Pg are bound together. The increase in t-PA activity is the result of a mechanism involving a t-PA lysine-dependent binding site in the GRP78 amino acid sequence (98)LIGRTWNDPSVQQDIKFL(115). We found that GRP78 is expressed on the surface of neuroblastoma SK-N-SH cells where it is co-localized with the voltage-dependent anion channel (VDAC), which is also a t-PA-binding protein in these cells. We demonstrate that both Pg and t-PA serve as a bridge between GRP78 and VDAC bringing them together to facilitate Pg activation. t-PA induces SK-N-SH cell proliferation via binding to GRP78 on the cell surface. Furthermore, Pg binding to the COOH-terminal region of GRP78 stimulates cell proliferation via its microplasminogen domain. This study confirms previous findings from our laboratory showing that GRP78 acts as a growth factor-like receptor and that its association with t-PA, Pg, and VDAC on the cell surface may be part of a system controlling cell growth.

The Inhibition of Human Tumor Cell Proliferation by RNase Pol, a Member of the RNase T1 Family, fromPleurotus ostreatus

RNase Po1 is a guanylic acid-specific ribonuclease (a RNase T1 family RNase) from Pleurotus ostreatus. We determined the cDNA sequence encoding RNase Po1 and expressed RNase Po1 in Escherichia coli. A comparison of the enzymatic properties of RNase Po1 and RNase T1 indicated that the optimum temperature for RNase Po1 activity was 20 °C higher than that for RNase T1. An MTT assay indicated that RNase Po1 inhibits the proliferation of human neuroblastoma cells (IMR-32 and SK-N-SH) and human leukemia cells (Jurkat and HL-60). Furthermore, Hoechst 33342 staining showed morphological changes in HL-60 cells due to RNase Po1, and flow cytometry indicated the appearance of a sub-G1 cell population. The extent of these changes was dependent on the concentration of RNase Pol. We suggest that RNase Po1 induces apoptosis in tumor cells.

Abstract 3128: Mxd3 is required for proliferation of human neuroblastoma cells.

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in children. Up to 45% of the cases are high risk at diagnosis, representing very aggressive, usually metastasized tumors that are refractory or recurrent after all available therapies. Prognosis for these patients is very poor with only 30% survival. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hedgehog (Hh) pathway (Yun, Rust, Ishimaru, Diaz, Mol Cell Biol, 2007) in granule neuron precursors. In these cells, overexpression of MXD3 results in higher MYCN expression, suggesting a functional interaction between the two transcription factors. MXD3 phenocopies MYCN expression in mouse models of medulloblastoma; we have also shown that it is expressed in human medulloblastoma and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS One, 2012). More than 20% of NBs have MYCN amplification, which is considered a marker of poor prognosis. In the current study, we investigated a possible role for MXD3 in NB cell proliferation. Using qRT-PCR we observed high levels of MXD3 mRNA expression in 5 NB cell lines: SK-N-DZ, IMR-32, SH-SY5Y, SK-N-BE and SK-N-SH. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in these cells. Furthermore, immunohistochemistry analysis showed strong immunoreactivity in a cohort of 16 primary NB tissue specimens. We investigated the role of MXD3 in NB cell proliferation by silencing MXD3 in the SK-N-DZ cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours, MXD3 protein levels were reduced &amp;gt;90% in cells infected with the shMXD3 virus but not with the control virus. MXD3 knockdown resulted in decreased proliferation in NB cells, supporting our hypothesis that it may be involved in the maintenance of NB. We analyzed cell cycle progression after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. Our results suggest that MXD3 is important for NB cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 is a potential candidate for targeted therapies against neuroblastoma. Citation Format: Gustavo Barisone, Noriko Satake, Carly Lewis, Connie Duong, Cathy Chan, Kit Lam, Jan Nolta, Elva Diaz. Mxd3 is required for proliferation of human neuroblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2013-3128

Abstract 2767: Nifurtimox and radiation for treating neuroblastoma: a preclinical study.

Abstract Neuroblastoma (NB) is the most common extracranial solid malignancy in infants and children. NB is responsible for more than 15% of deaths due to childhood cancers. It is an aggressive malignancy and often requires intensive multimodality treatment that potentially causes extensive long term side-effects. Although radiation therapy (XRT) is an important part of the standard treatment plan in this malignancy, XRT is known to cause significant side effects and morbidity associated with XRT greatly impacts the lives of many patients. Therefore, new approaches to increase the efficacy of XRT while decreasing side effects could have a great impact in the use of this therapy in cancer. XRT-induced cell death is partially associated with the generation of reactive oxygen species (ROS). ROS production is implicated in cell apoptosis, and induction of ROS potentially serves as a promising strategy for inhibiting tumor growth. In this investigation we exploited a strategy to increase the response of XRT by adding a chemical agent, Nifurtimox (Nfx) that is known to induce the generation of ROS. Human NB cells, SH-SY5Y and LA1-55n were treated with vehicle (DMSO) or increasing (5/7.5/10/20 μl/ml) concentrations of Nfx or XRT (1/2/5/10 Gy) and cell viability was determined at 24, 48 and 72 hr post-treatment. Both Nfx and XRT inhibited NB cell growth following a dose and time-dependent response. In order to test the combination response, these cells were treated with selected doses of Nfx and/or XRT and the cell viability was measured at 48 h post-treatment. The combination of both agents resulted in significantly higher inhibition when compared to single agent. ROS levels and apoptosis were assessed in NB cells following individual and combination treatment. ROS levels were measured using flow cytometry. Activation of caspases was determined by Caspase Glo kits while apoptotic cells were measured by flowcytometry (Annexin-V staining). Consistent with cell viability results, combination of XRT and Nfx significantly up-regulated ROS, caspases and induced apoptotic cell population when compared to individual treatment. For the in vivo assay, athymic nude mice were subcutaneously injected with LA1-55n cells. When tumor growth volume reached 100 mm3, mice were treated with Nfx (25 mg/kg/day in corn oil through oral gavage for 3 wk), XRT (5 Gy 2 time/wk for 2 wk) or both and the tumor growth was measured. At the termination of the experiment, combination therapy (XRT and Nfx) caused more than 80% tumor growth inhibition while the individual treatment (XRT or Nfx) caused approximately 60% inhibition of tumor growth. Results of this investigation demonstrate that the combination of XRT and Nfx synergistically inhibits human neuroblastoma cell proliferation and tumor growth in mice. Citation Format: Don Eslin, Chris M. Lee, Giselle S. Sholler, Umesh T. Sankpal, Robert M. Sutphin, Riyaz M. Basha. Nifurtimox and radiation for treating neuroblastoma: a preclinical study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2013-2767

Genistein inhibits estradiol- and environmental endocrine disruptor-induced growth effects on neuroblastoma cells in vitro

The aim of this study was to examine the effect of genistein on human neuroblastoma cell proliferation induced by two common environmental endocrine disruptors, bisphenol A (BPA) and Di-2-ethylhexyl phthalate (DEHP), and to investigate its underlying mechanism. SK-N-SH human neuroblastoma cells were treated with E2 (1 ng/ml), BPA (2 μg/ml) or DEHP (100 μM), with or without genistein (12.5 μM) in vitro. The number of viable cells was detected with an absorbance reader after 0, 24, 48 or 72 h treatment. The percentage of cells in different phases, and expression of Akt and its phosphorylation levels were also assessed by flow cytometry and western blot analysis at 72 h, respectively. The BPA and DEHP groups had a 30% higher number of viable cells compared to the non-treated group at 48 h (P<0.001). However, the cell numbers did not increase significantly in the groups with additional treatment with genistein (P>0.05 vs. control) and the same trend was observed at 72 h. The expression of phospho-Akt protein was increased in the groups treated with BPA or DEHP compared to the control group at 72 h (P<0.05), while no significant elevation in the expression of phospho-Akt was observed (P>0.05) in genistein-treated groups. Cells were arrested at the G2/M phase by genistein. Similar effects were observed in the E2 group with or without genistein treatment. Akt protein expression had no significant change among all the groups (P>0.05). In conclusion, estradiol- or environmental endocrine disruptor-induced proliferation of human neuroblastoma cells is effectively abolished by genistein, likely in a cell cycle- and Akt pathway-dependent manner.

Magnetic Shielding Accelerates the Proliferation of Human Neuroblastoma Cell by Promoting G1-Phase Progression

Organisms have been exposed to the geomagnetic field (GMF) throughout evolutionary history. Exposure to the hypomagnetic field (HMF) by deep magnetic shielding has recently been suggested to have a negative effect on the structure and function of the central nervous system, particularly during early development. Although changes in cell growth and differentiation have been observed in the HMF, the effects of the HMF on cell cycle progression still remain unclear. Here we show that continuous HMF exposure significantly increases the proliferation of human neuroblastoma (SH-SY5Y) cells. The acceleration of proliferation results from a forward shift of the cell cycle in G1-phase. The G2/M-phase progression is not affected in the HMF. Our data is the first to demonstrate that the HMF can stimulate the proliferation of SH-SY5Y cells by promoting cell cycle progression in the G1-phase. This provides a novel way to study the mechanism of cells in response to changes of environmental magnetic field including the GMF.

Reactive oxygen species(ROS) involving in inhibiting proliferation of human bone marrow neuroblastoma cells (SH-SY5Y) by giant typhonium rhizome water extract

Objective To investigate whether the reactive oxygen species involved in inhibiting proliferation of human bone marrow ceils of neuroblastoma cells （SH-SY5Y） process by giant typhonium rhizome water extract, and to investigate the initial mechanism of anti-tumor effect of giant typhonium rhizome in vitro. Methods After giant typhonium rhizome water extract processing of human bone marrow neuroblastoma cells （SH-SY5Y）at 24 h and 48 h, the cells were collected, and western blotting reaction methods such as flow cytometry was used to study the levels of ROS and SH-SY5Y apoptosis. Results The levels of ROS elevated fxom before dosing （4.9± 1.6）% to （39.7±11.8）% in the process of giant typhonium rhizome water extract induced apoptosis in SH-SY5Y; after ROS inhibition was added, SH-SY5Y apoptosis level induced by giant typhonium rhizome water extract had decreased. Conclusion ROS involved in the process of giant typhonium rhizome water extract inhibiting cell proliferation in SH-SY5Y. Key words: Giant typhonium rhizome; Reactive oxygen species; Human bone marrow neuroblastoma cells： Flow cytometry

Synergistic Effects of Combination Treatment with Bortezomib and Doxorubicin in Human Neuroblastoma Cell Lines

Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants. Currently, the mainstay of NB chemotherapy is combination treatment with some traditional drugs, but these combination regimens are always inefficient. Methods: The aim of this study was to evaluate the inhibitory effect of a combination of doxorubicin and bortezomib, a novel anticancer drug and the first prote-asome inhibitor approved for the treatment of human malignant tumors, on the proliferation of two human NB cell lines, SK-N-SH and SH-SY5Y. The general mechanism underlying this combined effect was also investigated. Synergistic inhibitory effects on human NB cell proliferation were evaluated using the median-effect principle. The pro-apoptotic effects of these drugs were evaluated using double staining with annexin-V-FITC and propidium iodide. Results: Synergistic inhibitory effects on proliferation were observed when a combination of bortezomib and doxorubicin was applied to cultured NB cells. A similar synergistic effect on apoptosis was also observed when the two drugs were used concurrently, which suggested that the possible mechanism underlying the observed synergistic inhibitory effect might be related to apoptosis. Conclusion: The combination of bortezomib and doxorubicin appears to be a promising strategy to treat NB.

Identification of potent c-Src inhibitors strongly affecting the proliferation of human neuroblastoma cells

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.

Abstract 3595: Abrogation of Akt signaling by Isobavachalcone contributes to its antiproliferative effect towards human cancer cells

Abstract The Akt (Protein kinase B, PKB) kinase is a key intermediate of many cellular processes and plays a critical role in cancer promotion and progression. Akt signaling pathway has attached much attention as a promising target for the development of cancer therapeutics. It was showed previously that Isobavachalcone (IBC), a natural chalcone, inhibits the proliferation of human neuroblastoma cells by causing cell apoptosis. However, the precise mechanisms of growth suppressive effects of IBC were not clear. The purpose of this study was to investigate the inhibitory activity of IBC on Akt signaling, which is probably involved in the proliferation inhibition induced by IBC. Here, the anti-proliferative activity of IBC was further verified in six human cancer cell lines, including ovarian carcinoma cell OVCAR-8, prostate carcinoma cell PC3, colorectal carcinoma cell colo205, breast carcinoma cell MCF-7, lung carcinoma cell A549 and hepatocellular carcinoma cell HepG2. Modeling results from the Sybyl/FlexiDock program suggested that IBC potentially bond to the ATP-binding pocket of Akt, which indicated that IBC might be a potential Akt inhibitor. Studies performed using the in vitro Akt kinase assay kit confirmed that IBC could directly target and inhibit Akt kinase in an ATP-competitive manner. Results from cell-based assays also revealed that IBC significantly abated Akt phosphorylation at Ser-473 and cellular Akt kinase activity, but did not interfere with Thr-308 phosphorylation of Akt. Consistent with Akt inhibition, IBC abrogated EGF-stimulated nuclear translocation of Akt as evaluated by fluorescence confocal microscopy. Cellular phosphorylation of Akt downstream substrates, including GSK3β, mTOR, p70S6K, 4E-BP1, XIAP and Bad, was also diminished by IBC. Consequently, significant levels of apoptosis were detected in IBC-treated cancer cells. Typical morphologic features of apoptosis, including chromatin condensation, nuclear fragmentation and formation of apoptotic bodies, were observed in IBC-treated cells by DAPI staining. Meanwhile, IBC also evoked cleavage of pro-caspase-9 and pro-caspase-3, poly (ADP-ribose) polymerase (PARP) degradation, up-regulation of reactive oxygen species (ROS) production and down-regulation of the ratio between Bcl-2 and Bax. Thus, IBC-induced apoptosis was associated with caspase- and ROS-involved mitochondria pathway. Taken together, we first reported IBC inhibited cancer proliferation by abrogation of Akt signaling, providing evidence for the further application of IBC as a novel therapeutic agent for human cancer therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3595.

Growth-promoting effect of bisphenol A on neuroblastoma in vitro and in vivo

Purpose To investigate the effect and mechanism of bisphenol A (BPA), one of the main environmental endocrine disruptors, on the proliferation of human neuroblastoma cells. Methods In vitro, cultured SK-N-SH cells were treated with 17 β-estradiol (E 2; 1 ng/mL), BPA (2 μg/mL) with or without estrogen receptor antagonist ICI182,780 (10 −6 mol/L). Viable cell number, DNA proliferation index, and expression of cyclin-dependent kinase 4 and cyclin D1 were assessed by absorbance reading, flow cytometry, and western blotting, respectively. In vivo, ovariectomized nude mice bearing SK-N-SH tumors were administered by gavage with E 2 (500 μg/kg per day, n = 11), BPA (200 mg/kg per day, n = 10), or vehicle (n = 9) for 18 days. Mice body weight, tumor volume and weight were examined every 3 days. Tumor microvessel density, proliferating cell nuclear antigen and vascular endothelial growth factor expression were evaluated by immunohistochemical staining or western blotting. Results In vitro, the BPA group had 20% higher number of viable cells, 70% higher proliferation index (both P < .01), and higher expression of cyclin-dependent kinase 4 and cyclin D1 than the nontreated group. In vivo, the BPA group had over 50% higher gross tumor volume, tumor weight, microvessel density, proliferating cell nuclear antigen ( P < .05 or .01), and higher vascular endothelial growth factor protein expression than the mock control group. Both in vitro and in vivo BPA effects were comparable with those by E 2. ICI182,780 effectively abolished the promoting effect for both. Conclusions Bisphenol A can promote the growth of neuroblastoma to a level similar to that of E 2. Estrogen receptor–dependent pathway and angiogenesis may contribute to the underlying mechanisms.

The interplay between microRNAs and the neurotrophin receptor tropomyosin-related kinase C controls proliferation of human neuroblastoma cells

MicroRNAs (miRNAs) are tiny noncoding RNAs whose function as modulators of gene expression is crucial for the proper control of cell growth and differentiation. Although the profile of miRNA expression has been defined for many different cellular systems, the elucidation of the regulatory networks in which they are involved is only just emerging. In this work, we identify a crucial role for three neuronal miRNAs (9, 125a, and 125b) in controlling human neuroblastoma cell proliferation. We show that these molecules act in an additive manner by repressing a common target, the truncated isoform of the neurotrophin receptor tropomyosin-related kinase C, and we demonstrate that the down-regulation of this isoform is critical for regulating neuroblastoma cell growth. Consistently with their function, these miRNAs were found to be down-modulated in primary neuroblastoma tumors.