• Journal of Korean Neurosurgical Society
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• v.57 no.3
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• pp.147-151
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• 2015

Objective : Neuroblastoma is one of common childhood tumors. Although its mortality is very high, there is no effective treatment yet. The aim of this project is to evaluate cytotoxic effects of melatonin (MLT) an endogen hormone and 13-cis retinoic acid (13-cis-RA) also named as isotretinoin an analogue of vitamin A on neuroblastoma SH-SY5Y cell line. Methods : In this study, SH-SY5Y cell line was used. After cell culture, the cells were exposed to different doses of MLT and 13-cis-RA. 24 and 48 hours later. While the viabilities was estimated with MTT cell viability assay test, apoptotic indexes were calculated after staining with TUNEL based apoptosis kit. Results : It was observed that MLT has very effective cytotoxic potential than 13-cis-RA on neuroblastoma cell line. At the same time, when MLT and 13-cis-RA were combined, this effect was potentiated. On the other hand, it was found that the effect of 13-cis-RA individually on neuroblastoma cells was very slight. Conclusion : We suggest that in the treatment of patient with neuroblastoma, MLT is very effective and also this effect can be augmented by combination with 13-cis-RA.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9445-9451
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• 2014

Neuroblastoma is the most common extracranial solid tumor in children. Approximately half of the affected patients are diagnosed with high-risk poor prognosis disease, and novel therapies are needed. Sanguinarine is a benzophenanthridine alkaloid which has anti-microbial, anti-oxidant and anti-inflammatory properties. The aim of this study is whether sanguinarine has in vitro apoptotic effects and which apoptotic genes might be affected in the human neuroblastoma cell lines SH-SY5Y (N-myc negative), Kelly (N-myc positive, ALK positive), and SK-N-BE(2). Cell viability was analysed with WST-1 and apoptotic cell death rates were determined using TUNEL. After RNA isolation and cDNA conversion, expression of 84 custom array genes of apoptosis was determined. Sanguinarine caused cell death in a dose dependent manner in all neuroblastoma cell lines except SK-N-BE(2) with rates of 18% in SH-SY5Y and 21% in Kelly human neuroblastoma cells. Cisplatin caused similar apoptotic cell death rates of 16% in SH-SY5Y and 23% in Kelly cells and sanguinarine-cisplatin combinations caused the same rates (18% and 20%). Sanguinarine treatment did not affect apoptototic gene expression but decreased levels of anti-apoptotic genes NOL3 and BCL2L2 in SH-SY5Y cells. Caspase and TNF related gene expression was affected by the sanguinarine-cisplatin combination in SH-SY5Y cells. The expression of regulation of apoptotic genes were increased with sanguinarine treatment in Kelly cells. From these results, we conclude that sanguinarine is a candidate agent against neuroblastoma.

• Journal of Acupuncture Research
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• v.19 no.1
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• pp.189-202
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• 2002

Objective : This study is designed to investigate the effects of bee venom and melittin on cell death in neuroblastoma cell line after pretreatment with NG(nerve growth inhibitory substance) Methods : It was evaluated by using MTT assay, morphological method, DNA fragmenation, flow cytometry, immunocytochemistry analysis, RT-PCR and Western blot. Results : The MTT assay demonstrated that neuroblastoma cell viability was significantly inhibited dose-dependently by treatment with bee venom and melittin after pretreatment with NG in comparison awith control. The morphological study and fow cytometry demonstrated that neuroblastoma cell showed apoptosis. DNA fragmenation showed DNA ladder below 1 Kbp. Immunocytochemistry assay demonstrated that Fos and MAPK were down-regulated. RT-PCR analysis demonstrated that Fos and MAPK was down-regulated. Western blot demonstrated that Fos and MAPK were down-regulated from $1{\mu}g/ml$ bee venom in neuroblastoma cell pretreated with NG. Conclusion : These result suggests that bee venom and melittin after NG treatment have significant anti-cancer effect and further study is needed in vivo.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.18
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• pp.8313-8319
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• 2016

We have recently shown that thymoquinone (TQ) has a potent cytotoxic effect and induces apoptosis via caspase-3 activation with down-regulation of XIAP in mouse neuroblastoma (Neuro-2a) cells. Interestingly, our results showed that TQ was significantly more cytotoxic towards Neuro-2a cells when compared with primary normal neuronal cells. In this study, the effects of TQ on cell-cycle regulation and the mechanisms that contribute to this effect were investigated using Neuro-2a cells. Cell-cycle analysis performed by flow cytometry revealed cell-cycle arrest at G2/M phase and a significant increase in the accumulation of TQ-treated cells at sub-G1 phase, indicating induction of apoptosis by the compound. Moreover, TQ increased the expression of p53, p21 mRNA and protein levels, whereas it decreased the protein expression of PCNA, cyclin B1 and Cdc2 in a dose-dependent manner. Our finding suggests that TQ could suppress cell growth and cell survival via arresting the cell-cycle in the G2/M phase and inducing apoptosis of neuroblastoma cells.

• Journal of Acupuncture Research
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• v.20 no.2
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• pp.98-111
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• 2003

Objective : This study was designed to analyze the effects of bee venom and melittin on cell death in neuroblastoma cell line. Methods : MTT assay, morphologic method, DNA fragmentation, flow cytometry, immunocytochemistry analysis, RT-PCR and Western blot were performed. Results : The obtained results are summarized as follows: 1. The MTT assay demonstrated that neuroblastoma cell viability was significantly inhibitted dose-dependently by treatment with bee venom and melittin in comparison with control. 2. Cell culture demonstrated that control group proliferated highestly at he 5th day in comparison with the 4th day in bee venom and melittin group. And in bee venom and melitti group cell proliferation decreased 2.5 times than control group. 3. The morphologic study demonstrated that neuroblastoma cell showed apoptosis after treatment with bee venom and melittin for 6 hours using microscope. 4. The Flow cytometry demonstrated that apoptosis of neuroblastoma cell treated with bee venom and melittin was related with stop of cell cycle in stage of $G_0/G_1$. 5 .DNA fragmenation demonstrated that neuroblastoma cell treated with bee venom and melittin showed DNA ladder below 1 Kbp. 6. Immunocytochemistry assay demonstrated that Fos and MAPK which are related with cancer were down-regulated by treatment with bee venom and melittin. 7. RT-PCR analysis demonstrated that Fos and MAPK mRNA were transcripted. Fos was down-regulated form treatment with $5{\mu}g/ml$ bee venom and MAPK was down-regulated form $1{\mu}g/ml$ bee venom. 8. Western blot demonstrated that Fos was down-regulated from $1{\mu}g/ml$ bee venom whereas MAPK was expressed by $1{\mu}g/ml$ bee venom but down-regulated by $10{\mu}g/ml$ bee venom. Conclusions : We found that some cancer related genes ware down-regulated by treatment with bee venom and melittin. Further study is needed for investigating the anti-cancer effect of bee venom and melittin.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.51-54
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• 1998

It has been proved that co-cultivation of human neroblastoma cells and human fibroblast cells can enhance nerve cell growth and the production of BDNF in perfusion cultivation. In batch co-cultivation, maximum cell density was increased up to 1.76${\times}$106 viable cells/mL from 9${\times}$105 viable cells/mL of only neuroblastoma cell culture. The growth of neuroblastoma cells was greatly improved by culturing both nerve and fibroblast cells in a perfusion process, maintaining 1.5${\times}$106 viable cells/mL, which was much higher than that form fed-batch cultivation. The nerve cell growth was greatly enhance in both fed-batch and perfusion cultivations while the growth of fibroblast cells was not. It strongly implies that the factors secreted from human fibrobast cells and/or the environments of co-culture system can enhance both cell growth and BDNF secretion. Specific BDNF production rate was not enhanced in co-cultures; however, the production period was increased as the cell growth was lengthened in the co-culture case. Competitive growth between nerve cells and fibroblast cells was not observed in all cases, showing no changes of fibroblast cell growth and only enhancement of the neuroblastoma cell growth and overall BDNF production. It was also found that the perfusion cultivation was the most appropriate process for cultivating two cell lines simultaneously in a bioreactor.

• The Korean Journal of Physiology and Pharmacology
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• v.2 no.4
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• pp.411-417
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• 1998

The role of phospholipase ($A_2\;PLA_2$) in tumor cell growth was investigated using SK-N-MC human neuroblastoma cells. 4-Bromophenacyl bromide (BPB) and mepacrine (Mep), known $PLA_2$ inhibitors, suppressed growth of the tumor cells in a dose-dependent manner without a significant cytotoxicity. Melittin (Mel), a $PLA_2$ activator, enhanced the cell growth in a concentration-dependent fashion. The growth-enhancing effects of Mel were significantly reversed by the co-treatment with $PLA_2$ inhibitors. In addition, Mel induced intracellular $Ca^{2+}$ release from internal stores like as did serum, a known intracellular $Ca^{2+}$ agonist in the tumor cells. Intracellular $Ca^{2+}$ release induced by these agonists was significantly blocked by $PLA_2$ inhibitors at growth-inhibitory concentrations. Arachidonic acid (AA), a product of the $PLA_2-catalyzed$ reaction, induced cell growth enhancement and intracellular $Ca^{2+}$ release. These effects of AA were significantly blocked by BAPTA/AM, an intracellular $Ca^{2+}$ chelator. Taken together, these results suggest that the modulation of $PLA_2$ activity may be one of the regulatory mechanisms of cell growth in human neuroblastoma cells. Intracellular $Ca^{2+}$ may act as a key mediator in the $PLA_2-induced$ growth regulation.

• The Journal of Korean Medicine
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• v.26 no.4
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• pp.12-21
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• 2005

Objectives: In the present study, we investigated whether an aqueous extract of Armeniacae semen induces apoptotic neuronal cell death upon mouse N2a neuroblastoma cells. Methods: 1. Cell viability was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTI) assay. 2. For in situ detection of apoptotic cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, 4,6-diamidino-2-phenylindole (DAPI) staining. 3. The fraction of cells was revealed by flow cytometric analysis used that. 4. For detection of apoptotic DNA cleavage, DNA fragmentation assay was performed. 5. For detection of bax and bcl-2, Western blot analysis was performed. 6. Caspase enzyme activity was measured using caspase-3 assay. Results: From the present results, N2a neuroblastoma cells treated with Armeniacae semen extract exhibited several characteristics of apoptosis. A treatment of Armeniacae semen extract was shown to increase the expression of Bax, a proapoptotic protein, and the treatment decreased the expression of Blc2, an anti-apoptotic protein. In addition, Armeniacae semen extract increased the caspase-3 enzyme activity. Conclusions: The present results show that Armeniacae semen extract induces apoptotic cell death in mouse N2a neuroblastoma cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4671-4676
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• 2014

Background: MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours but little is known about its role in neuroblastoma. Our aim was to investigate the potential role and mechanism of miR-200a in neuroblastomas. Materials and Methods: Expression levels of miR-200a in tissues were determined using RT-PCR. The effect of miR-200a and shAP-$2{\gamma}$ on cell viability was evaluated using MTS assays, and target protein expression was determined using Western blotting and RT-PCR. Luciferase reporter plasmids were constructed to confirm direct targeting. Results were reported as mean${\pm}$S.E.M and differences were tested for significance using the 2-tailed Students t-test. Results: We determined that miR-200a expression was significantly lower in neuroblastoma tumors than the adjacent non-cancer tissue. Over-expression of miR-200 are reduced cell viability in neuroblastoma cells and inhibited tumor growth in mouse xenografts. We identified AP-$2{\gamma}$ as a novel target for miR-200a in neuroblastoma cells. Thus miR-200a targets the 3'UTR of AP-$2{\gamma}$ and inhibits its mRNA and protein expression. Furthermore, our result showed that shRNA knockdown of AP-$2{\gamma}$ in neuroblastoma cells results in significant inhibit of cell proliferation and tumor growth in vitro, supporting an oncogenic role of AP-$2{\gamma}$ in neuroblastoma. Conclusions: Our study revealed that miR-200a is a candidate tumor suppressor in neuroblastoma, through direct targeting of AP-$2{\gamma}$. These findings re-enforce the proposal of AP-$2{\gamma}$ as a therapeutic target in neuroblastoma.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.239-244
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• 2014

Several recent studies have showed that the n-myc downstream regulated gene 2 (NDRG2) is a new tumor suppressor gene, and that it plays an important role in tumor suppression in several cancers or cancer cell lines. However, few studies focused on its function in neuroblastoma cells. In the present investigation, we demonstrated that NDRG2 overexpression inhibited their proliferation. Using a cDNA microarray, we found that overexpression of NDRG2 inhibited the expression of cysteine-rich protein 61 (CYR61), a proliferation related gene. From our research, CYR61 may partially hinder NDRG2-mediated inhibition of cell proliferation. Overexpression of NDRG2 resulted in accumulation of cells in the G1 phase, which was accompanied by upregulation of p21 and p27 and downregulation of CDK4 and cyclin D1. Taken together, these data indicate that NDRG2 inhibits the proliferation of neuroblastoma cells partially through suppression of CYR61. Our findings offer novel insights into the physiological roles of NDRG2 in neuroblastoma cell proliferation, and NDRG2 may prove to be effective candidate for the treatment of children with neuroblastoma.