Human Neuroblastoma Cell Line Research Articles

DUSP5 expression associates with poor prognosis in human neuroblastoma

Regulation of growth and differentiation of neuroblastoma (NB) cells is the rational of some maintenance therapies for high-risk NB. MAP kinase phosphatases (MKPs) are potential physiologic regulators of neuronal differentiation and survival, but their expression patterns in NB are scarcely known. Here, an expression analysis of the MKP family has been performed using human NB tumor samples and human NB cell lines (SH-SY5Y, SMS-KCNR, and IMR-32) undergoing retinoic acid (RA)-induced differentiation or subjected to stimuli that activate the MAPK ERK1/2 pathway. We have identified candidate MKPs that could modulate differentiation and growth of NB cells. pERK1/2 high expression correlated with high expression of the MKP DUSP5 in NB tumors, and was associated with poor prognosis. ERK1/2 activation on SH-SY5Y cells was accompanied by increased cell proliferation, and correlated with the expression levels of DUSP5. Accordingly, siRNA knock-down of DUSP5 augmented proliferation of SH-SY5Y cells. Our findings provide insights into the dynamic expression of MKPs in NB cells, disclose DUSP5 as a potential marker of NB poor prognosis, and suggest a role for DUSP5 in limiting ERK1/2-mediated NB proliferation.

Ellagic acid: Insights into its neuroprotective and cognitive enhancement effects in sporadic Alzheimer's disease

Sporadic Alzheimer's disease (SAD), an age-associated dementia, is described as neuronal loss and marked cognitive impairment. Ellagic acid (EA) is a phenolic phytoconstituent obtained from grains and fruits, having evident antioxidant effects and known to modulate several endogenous molecular signals in humans in a beneficial way. The current study evaluated the safety profile of EA in the SH-SY5Y human neuroblastoma cell line, performing anti-oxidative effect by DPPH assay, and evaluating anti-AchE (acetylcholinesterase) effect against AchE enzyme from Electrophorus electricus. The observations were further confirmed by in vivo therapeutic effects in streptozotocin (STZ)-induced SAD rats in the context of altered biochemical and behavioral features. Treatment with EA (50 mg/kg, p.o.) for 30 days revealed reduction in STZ (3 mg/kg i.c.v.) prompted SAD and associated biochemical abnormalities in experimental rats which included diminished oxidative stress profile, pro-inflammatory markers i.e. GFAP and CRP; AchE level, and amyloid-β plaque level. Moreover, an elevated level of synaptophysin indicated improved synaptic connectivity, and intact neural architecture showed neuroprotection in the EA group. Furthermore, the behavioral investigation by maze paradigms revealed reduced locomotor behavior, irregular spontaneous alternation, declination in memory score and increased memory errors in SAD rats. EA treatment normalized these SAD-associated abnormal behavioral representations in rats. Hence our findings suggest neuroprotective effects of EA and improvement in cognitive behavior in SAD rats.

Reduction of PINK1 or DJ-1 impair mitochondrial motility in neurites and alter ER-mitochondria contacts.

Subcellular distribution of mitochondria in neurons is crucial for meeting the energetic demands, as well as the necessity to buffer Ca2+ within the axon, dendrites and synapses. Mitochondrial impairment is an important feature of Parkinson disease (PD), in which both familial parkinsonism genes DJ‐1 and PINK1 have a great impact on mitochondrial function. We used differentiated human dopaminergic neuroblastoma cell lines with stable PINK1 or DJ‐1 knockdown to study live motility of mitochondria in neurites. The frequency of anterograde and retrograde mitochondrial motility was decreased in PINK1 knockdown cells and the frequency of total mitochondrial motility events was reduced in both cell lines. However, neither the distribution nor the size of mitochondria in the neurites differed from the control cells even after downregulation of the mitochondrial fission protein, Drp1. Furthermore, mitochondria from PINK1 knockdown cells, in which motility was most impaired, had increased levels of GSK3βSer9 and higher release of mitochondrial Ca2+ when exposed to CCCP‐induced mitochondrial uncoupling. Further analysis of the ER‐mitochondria contacts involved in Ca2+ shuttling showed that PINK1 knockdown cells had reduced contacts between the two organelles. Our results give new insight on how PINK1 and DJ‐1 influence mitochondria, thus providing clues to novel PD therapies.

Curcumin-loaded self-nanomicellizing solid dispersion system: part II: in vivo safety and efficacy assessment against behavior deficit in Alzheimer disease.

Curcumin (CUR), a natural polyphenolic compound, is considered as one of the most potential candidates against Alzheimer disease (AD) by targeting multiple pathologies such as amyloid-beta, tau phosphorylation, and oxidative stress. Poor physicochemical profile and oral bioavailability (BA) are the major contributors to its failure in clinical trials. Lack of success in numerous drug clinical trials for the treatment of AD urges the need of repositioning of CUR. To overcome its limitation and enhance oral BA, Novel CUR Formulation (NCF) was developed using self-nanomicellizing solid dispersion strategy which displayed 117-fold enhancement in oral BA of CUR. NCF was tested using SH-SY5Y695 APP human neuroblastoma cell line against the cytotoxicity induced by copper metal ion, H2O2, and Aβ42 oligomer and compared with CUR control. The safety and efficacy of NCF on mice AD-like behavioral deficits (open field, novel objective recognition, Y-maze, and Morris water maze tests) were assessed in transgenic AD (APPSwe/PS1deE9) mice model. In SH-SY5Y695 APP human neuroblastoma cell line, NCF showed better safety and efficacy against the cytotoxicity due to the significantly enhancement of cellular uptake. It not only prevents the deterioration of cognitive functions of the aged APPSwe/PS1deE9 mice during aging but also reverses the cognitive functions to their much younger age which is also better than the currently available approved options. Moreover, NCF was proved as well tolerated with no appearance of any significant toxicity via oral administration. The results of the study demonstrated the potential of NCF to improve the efficacy of CUR without compromising its safety profile, and pave the way for clinical development for AD.

Exogenous expression of miRNA-3613-3p causes APAF1 downregulation and affects several proteins involved in apoptosis in BE(2)-C human neuroblastoma cells

MicroRNAs (miRNAs) are a class of small non‑coding RNAs involved in post‑transcriptional gene regulation. Furthermore, dysregulation of miRNA expression is an important factor in the pathogenesis of neuroblastoma. Our previous study identified that overexpression of monocyte chemoattractant protein‑induced protein1 protein led to a significant downregulation of a novel miRNA molecule, miRNA‑3613‑3p. In the present study, the potential involvement of miRNA‑3613‑3p in the cell biology of neuroblastoma was investigated. It was identified that the expression of miRNA‑3613‑3p varies among a range of human neuroblastoma cell lines. As the delineation of the functions of a miRNA requires the identification of its target genes, seven putative mRNAs that may be regulated by miRNA‑3613‑3p were selected. Furthermore, it was identified that overexpression of miRNA‑3613‑3p causes significant downregulation of several genes exhibiting tumor suppressive potential [encoding apoptotic protease‑activating factor1(APAF1), Dicer, DNA fragmentation factor subunitβ, vonHippel‑Lindau protein and neurofibromin1] in BE(2)‑C human neuroblastoma cells. APAF1 mRNA was the most significantly decreased transcript in the cells with miRNA‑3613‑3p overexpression. In accordance with the aforementioned results, the downregulation of cleaved caspase-9 and lack of activation of executive caspases in BE(2)‑C cells following miRNA‑3613‑3p overexpression was observed. The results of the present study suggest a potential underlying molecular mechanism of apoptosis inhibition via APAF1 downregulation in human neuroblastoma BE(2)‑C cells with miRNA‑3613‑3p overexpression.

Schwann cells protect against CaMKII- and PKA-dependent Acrylamide-induced Synapsin I phosphorylation

ObjectivesTo explore the effects of Acrylamide (ACR), as well as the influence of Schwann cells (SCs), on the signal transduction pathway and phosphorylation of Synapsin I in a Human neuroblastoma cell line (NB-1). MethodsNB-1s, NB-1s co-cultured with SCs, and a negative control group (NB-1 cells without ACR) were exposed to gradient concentrations of ACR for 48 h. Cell proliferation and viability were determined by MTT. Protein and mRNA expression levels of typical kinases (i.e., cAMP-dependent protein kinase [PKA], calcium/calmodulin-dependent protein kinase II [CaMKII], and mitogen-activated protein kinase-extracellular signal-regulated kinases [MAPK-Erk]), their phosphorylation status, as well as Synapsin I and its phosphorylation status, were tested by western blotting and polymerase chain reaction, respectively. Further, the effect of SCs on ACR-induced NB-1 cell toxicity was evaluated. Results(1) The MTT assay showed a sustained, dose- and time-dependent inhibition of NB-1s exposed to ACR. (2) ACR exposure increased the phosphorylation of CaMKII and PKA, which subsequently increased the phosphorylation of Synapsin I (at Serine603 [a substrate site of CaMKII] and Serine9 [a substrate site of PKA]). Pretreatment with CaMKII and PKA inhibitors blocked the ACR-mediated increase in phosphorylation. The above-described results were all significantly different when compared to the control group (p < 0.05). (3) When co-cultured with SCs, ACR-induced NB-1 inhibition was obviously decreased, and the trend of change of phosphorylated CaMKII, PKA, and Synapsin I were changed (first slightly increased and then decreased), which was inconsistent with what we observed in NB-1s cultured alone. ConclusionsThe toxic effects of ACR on neurons may be mediated by CaMKII and PKA-dependent signaling pathways in which Synapsin I may act as a downstream effector. Furthermore, glial cells (SCs) may be able to prevent a certain degree of ACR-induced neuronal damage.

Proteomic Changes in Chick Brain Proteome Post Treatment with Lathyrus Sativus Neurotoxin, \u03b2-N-Oxalyl-L-\u03b1,\u03b2-Diaminopropionic Acid (L-ODAP): A Better Insight to Transient Neurolathyrism

Neurolathyrism is a neurodegenerative disorder characterized by spastic paraplegia resulting from the excessive consumption of Lathyrus sativus (Grass pea). β-N-Oxalyl-L-α,β-diaminopropionic acid (L-ODAP) is the primary neurotoxic component in this pea. The present study attempted to evaluate the proteome-wide alterations in chick brain 2 hr and 4 hr post L-ODAP treatment. Proteomic analysis of chick brain homogenates revealed several proteins involved in cytoskeletal structure, signaling, cellular metabolism, free radical scavenging, oxidative stress and neurodegenerative disorders were initially up-regulated at 2 hr and later recovered to normal levels by 4 hr. Since L-ODAP mediated neurotoxicity is mainly by excitotoxicity and oxidative stress related dysfunctions, this study further evaluated the role of L-ODAP in apoptosis in vitro using human neuroblastoma cell line, IMR-32. The in vitro studies carried out at 200 μM L-ODAP for 4 hr indicate minimal intracellular ROS generation and alteration of mitochondrial membrane potential though not leading to apoptotic cell death. L-ODAP at low concentrations can be explored as a stimulator of various reactive oxygen species (ROS) mediated cell signaling pathways not detrimental to cells. Insights from our study may provide a platform to explore the beneficial side of LODAP at lower concentrations. This study is of significance especially in view of the Government of India lifting the ban on cultivation of low toxin Lathyrus varieties and consumption of this lentil.

Cdc45/Mcm2-7/GINS complex down-regulation mediates S phase arrest in okadaic acid-induced cell damage

Okadaic acid (OA) is one of the most common and widespread marine toxins and causes acute gastrointestinal symptoms known as diarrheic shellfish poisoning (DSP) in humans. Although OA is not classified as a typical neurotoxin, an increasing number of studies have reported its neurotoxic effects. However, most of the available studies have focused on OA-induced inhibition of serine/threonine protein phosphatases, while the molecular mechanism of OA-induced neurotoxicity remains largely unclear. To better understand the potentially toxicological profile of OA, cell cycle arrest, DNA damage and alterations in gene expression in the human neuroblastoma cell line SHSY5Y upon OA exposure were determined using flow cytometry, comet assay, and transcriptome microarray. The results showed that OA could induce cell cycle arrest at S phase and might be involved in significant DNA strand breaks. Gene expression profiling indicated that the differentially expressed genes after OA exposure were significantly enriched in the “DNA replication” and “cell cycle” pathways. Real-time PCR result had further validated that down-regulation of the Cdc45/Mcm2-7/GINS complex might be the major factor regulating those alterations. These findings provide new insight into the molecular mechanisms of OA-induced neurotoxicity, and the current data may also provide a basis for future studies.

In vitro neuroprotective effects of naringenin nanoemulsion against β-amyloid toxicity through the regulation of amyloidogenesis and tau phosphorylation

Alzheimer's disease (AD) is an increasingly prevalent neurological disorder of the central nervous system. There is growing evidence that amyloidogenesis is a pathological hallmark for AD; this leads to the formation of senile plaques. Naringenin is a bioflavonoid which has neuroprotective effects through its antioxidant and anti-inflammatory properties. However, its clinical usage is limited due to its inefficient transport across biological membranes. In the present study, a naringenin nanoemulsion was prepared and its neuroprotective effects were tested against β-amyloid induced neurotoxicity in a human neuroblastoma cell line (SH-SY5Y). The optimised, naringenin-loaded nanoemulsion formulation had a droplet size of 113.83 ± 3.35 nm and around 50 nm, as assessed respectively by photon correlation spectroscopy and transmission electron microscopy. The preparation showed a low polydispersity index (0.312 ± 0.003), a high zeta potential (12.4 ± 1.05) and a high percentage transmittance (97.01%). The neuroprotective activity of naringenin nanoemulsions was determined by assessing their ability to protect SH-SY5Y neuroblastoma cells against the neurotoxic effect of beta amyloid (Aβ). Aβ-induced production of reactive oxygen species (ROS), amyloid precursor protein (APP), β-secretase (BACE), total tau and phosphorylated tau (pT231) was also determined. The naringenin loaded nanoemulsion significantly alleviated the direct neurotoxic effects of Aβ on SH-SY5Y cells; this was associated with a down-regulation of APP and BACE expression, indicating reduced amyloidogenesis. Furthermore, it decreased the levels of phosphorylated tau in SH-SY5Y cells exposed to Aβ. These results suggest that a naringenin-loaded nanoemulsion could be a promising agent for the treatment of Alzheimer's disease.

Abstract 3177: mTOR inhibitor augments the antitumor effect of p53-expressing oncolytic adenovirus in human neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB patients show an unfavorable clinical outcome, even after treatment with intensive chemotherapy and aggressive surgical resection. Therefore, a novel therapeutic strategy is needed for the treatment of high-risk NB patients. We developed a tumor-specific replication-competent oncolytic adenovirus OBP-702, in which the hTERT promoter drives the expression of the viral E1A and E1B genes for tumor-specific viral replication and the Egr-1 promoter induces p53 expression. Preclinical studies demonstrated that OBP-702 induces autophagy-related cell death in human NB cell lines. To enhance OBP-702-induced autophagy-related cell death, we hypothesized that a mTOR inhibitor temsirolimus, which is well-known autophagy inducer, may promote autophagy-related cell death by OBP-702 infection. In this study, we investigated the antitumor effect of OBP-702 in combination with temsirolimus in human NB cell lines. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The antitumor effect of OBP-702 and temsirolimus was evaluated using XTT assay. In vitro synergistic effect was calculated with CalcuSyn software (BioSoft, Inc.). Virus-mediated cell death and p53 expression were analyzed by Western blot analysis. Ad-p53, a replication-defective adenovirus expressing p53 gene, was used to assess the effect of p53 in combination with temsirolimus. The combined effect of OBP-702 and temsirolimus was further evaluated using a subcutaneous CHP-134 xenograft tumor model. Results: Combination with OBP-702 and temsirolimus exhibited a synergistic antitumor effect in all NB cell lines in vitro. We confirmed that combination with these drugs enhanced autophagy. In addition, temsirolimus showed an effect to stabilize p53 protein induced by OBP-702. Ad-p53 also showed synergistic effect in combination with temsirolimus. The combination treatment significantly inhibited tumor growth, as compared to monotherapy, in NB xenograft tumor model. Conclusions: These results suggest that p53-expressing oncolytic adenovirus and mTOR inhibitor synergistically induce autophagic cell death and may be a promising strategy in NB patients. Citation Format: Terutaka Tanimoto, Hiroshi Tazawa, Hiroshi Noso, Morimichi Tani, Takanori Oyama, Yasuo Urata, Shunsuke Kagawa, Takuo Noda, Toshiyoshi Fujiwara. mTOR inhibitor augments the antitumor effect of p53-expressing oncolytic adenovirus in human neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3177.

New Targets for Parkinson's Disease: Adhesion G Protein-Coupled Receptor B1 is Downregulated by AMP-Activated Protein Kinase Activation.

While progressive dopaminergic neurodegeneration is responsible for the cardinal motor defects in Parkinson's disease (PD), new diagnostics and therapeutic targets are necessary to effectively address this major global health burden. We evaluated whether the adhesion G protein-coupled receptor B1 (ADGRB1, formerly BAI1, brain-specific angiogenesis inhibitor 1) might contribute to dopaminergic neuronal loss. We used bioinformatic analyses, as well as in vitro and in vivo PD models. We report in this study that ADGRB1 is decreased in PD and that the ADGRB1 level is specifically decreased in dopaminergic neurons in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In primary mouse mesencephalic neurons and human neuroblastoma cell lines, 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of MPTP, suppressed the expression of ADGRB1. Moreover, we applied a network generation tool, Ingenuity Pathway Analysis®, with the transcriptomics dataset to extend the upstream regulatory pathway of ADGRB1 expression. AMP-activated protein kinase (AMPK) was predicted as a regulator, and consequently, 5-aminoimidazole-4-carboxamide ribonucleotide, a specific activator of AMPK, reduced the ADGRB1 protein level. Finally, ADGRB1 overexpression decreased nuclear condensation induced by MPP+ treatment. Taken together, we observed that decreased ADGRB1 by activation of AMPK induced neuronal cell death in MPTP/MPP+-mediated PD models, suggesting that ADGRB1 might potentially play a survival role in the neurodegenerative pathway of PD. These data offer new insights into dopaminergic cell death with therapeutic implications for neurodegenerative disorders.

Self-nanomicellizing solid dispersion of edaravone: part II: in vivo assessment of efficacy against behavior deficits and safety in Alzheimer's disease model.

BackgroundAlzheimer’s disease (AD) is a devastating neurodegenerative disorder that lacks any disease-modifying drug for the prevention and treatment. Edaravone (EDR), an approved free radical scavenger, has proven to have potential against AD by targeting multiple key pathologies including amyloid-beta (Aβ), tau phosphorylation, oxidative stress, and neuroinflammation. To enable its oral use, novel edaravone formulation (NEF) was previously developed. The aim of the present investigation was to evaluate safety and efficacy of NEF by using in vitro/in vivo disease model.Materials and methodsIn vitro therapeutic potential of NEF over EDR was studied against the cytotoxicity induced by copper metal ion, H2O2 and Aβ42 oligomer, and cellular uptake on SH-SY5Y695 amyloid-β precursor protein (APP) human neuroblastoma cell line. For in vivo safety and efficacy assessment, totally seven groups of APP/PS1 (five treatment groups, one each as a basal and sham control) and one group of C57BL/6 mice as a positive control for behavior tests were used. Three groups were orally treated for 3 months with NEF at an equivalent dose of EDR 46, 138, and 414 µmol/kg, whereas one group was supplied with each Donepezil (5.27 µM/kg) and Soluplus (amount present in NEF of 414 µmol/kg dose of EDR). Behavior tests were conducted to assess motor function (open-field), anxiety-related behavior (open-field), and cognitive function (novel objective recognition test, Y-maze, and Morris water maze). For the safety assessment, general behavior, adverse effects, and mortality were recorded during the treatment period. Moreover, biochemical, hematological, and morphological parameters were determined.ResultsCompared to EDR, NEF showed superior cellular uptake and neuroprotective effect in SH-SY5Y695 APP cell line. Furthermore, it showed nontoxicity of NEF up to 414 µM/kg dose of EDR and its potential to reverse AD-like behavior deficits of APP/PS1 mice in a dose-dependent manner.ConclusionOur results indicate that oral delivery of NEF holds a promise as a safe and effective therapeutic agent for AD.

PO-207 The role of RSU-1 in glioma cells metastasis

IntroductionRas Suppressor-1 (RSU-1) was recently found to be associated with Focal Adhesion (FA) proteins. The main objective of this research work was the in vitrocharacterisation of a panel of glioma cancer cells in terms of aggressiveness, and the investigation of RSU-1 role on invasion of glioma cancer cells.Material and methodsA panel of four human neuroblastoma cell lines was used (H4, SW1088, A172, U87-MG). Invasion assay with matrigel-coated transwell and soft agar growth assay were performed in order to characterise the aggressiveness of glioma cell lines. The RSU-1 expression for glioma cells was tested by real time PCR and immunoblotting. Finally, glioma cells were transfected with siRNA against RSU-1 in order to find out the role of RSU-1 in glioma cells.Results and discussionsIn order to assess the invasive potential of the four glioma cell lines, a transwell invasion assay was performed and A172 and U87-MG cells found to be more invasive than H4 and SW1088 cells. To determine the aggressiveness of the studied cell lines, soft agar assay was also performed. The number of colonies for the U87-MG and A172 was significantly larger than the H4 and SW1088, with the latter cell lines only forming a few small colonies.We then sought to find out whether RSU-1 gene is differentially expressed in the four cell lines and whether its expression is correlated with invasiveness. It was found that the more aggressive A172 and U87-MG cell lines, overexpress RSU-1 compared to the less aggressive H4 and SW1088 cell lines.Subsequently, two cell lines were selected to be used for further experiments, H4 and A172 which are the least aggressive and more aggressive cells, respectively. Our results show that upon RSU-1 silencing, the invasion of A172 cells was significantly decreased whereas invasion of H4 cells was increased with respective changes observed in the expression of Matrix Metalloproteinase 13 (MMP13), a fundamental protease in cancer cell metastasis.ConclusionResults confirmed that the A172 and U87-MG glioma cells are more aggressive than H4 and SW1088. Also, RSU-1 was found to be overexpressed in most aggressive cells in comparison to less aggressive cell lines. More aggressive A172 cells lacking RSU-1 showed decreased invasion while H4 cells showed increased invasion. Collectively, RSU-1 found to be critical for glioma cell invasion and further investigation of the implicated molecular mechanism is underway.

Abstract 3831: Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma

Abstract Background: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. Despite the recent advances in the treatment of NB, patients with high-risk NB still have poor prognosis. Immunotherapy using anti-GD2 monoclonal antibody (mAb), which binds to disialoganglioside GD2 on the surface of NB cells, has recently become a standard treatment for high-risk NB. However, the therapeutic efficacy of anti-GD2 mAb is insufficient in many clinical trials. Therefore, the enhancement of therapeutic potential of anti-GD2 mAb is needed to improve the clinical outcome of patients with high-risk NB. Near-infrared photoimmunotherapy (NIR-PIT) is a promising antitumor strategy to enhance the therapeutic potential of immunotherapy using an antibody-photoabsorber conjugate (APC). In this study, we investigated the therapeutic efficacy of anti-GD2-based NIR-PIT using anti-GD2 mAb conjugated to the photoabsorber IR700 (anti-GD2-IR700) in human NB cells. Methods: We used 3 human NB cell lines, including IMR-32, CHP-134 and SK-N-SH. The expression of GD2 on the cell surface of NB cells was analyzed by flow cytometric analysis. Anti-GD2-IR700 was synthesized by incubating anti-GD2 mAb and IR700. The mixture was purified with a Sephadex G50 column. The antitumor effect of anti-GD2-IR700 was evaluated using XTT assay. NB cells were incubated with anti-GD2-IR700, anti-GD2 mAb or PBS and NIR light was irradiated at 2.0J/cm2. Results: IMR-32 and CHP-134 cells, but not SK-N-SH cells, expressed GD2 protein on the cell surface. In GD2-positive IMR-32 and CHP-134 cells, administration of anti-GD2-IR700 significantly suppressed the cell viability compared to anti-GD2 mAb or PBS when combined with NIR light irradiation. In contrast, in GD2-negative SK-N-SH cells, anti-GD2-IR700 showed no significant inhibition of cell viability in combination with NIR light irradiation. Conclusion: These results suggest that NIR-PIT using an anti-GD2-IR700 is a promising antitumor strategy to promote the therapeutic efficacy of anti-GD2 immunotherapy for high-risk NB. Citation Format: Hiroshi Nouso, Hiroshi Tazawa, Terutaka Tanimoto, Morimichi Tani, Takanori Oyama, Hiroaki Sato, Kazuhiro Noma, Shunsuke Kagawa, Hisataka Kobayashi, Takuo Noda, Toshiyoshi Fujiwara. Development of near-infrared photoimmunotherapy targeting GD2-positive neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3831.

Abstract 1902: Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma

Abstract The purpose of this study was to determine the effect of the natural product Harmine in human neuroblastoma (NB). NB is an early childhood malignancy arising from the developing peripheral nervous system. In up to 25% of cases MYCN gene amplification is correlated to high tumor stage and poor patient prognosis. High stage NB relapses frequently in spite of multimodal therapy and is then virtually untreatable. There is a clear need for specific, novel therapeutics. Harmine is a tricyclic β-carboline alkaloid from the harmal plant with cytostatic and cytotoxic effects on tumor cells. It is capable of blocking the activity of mitogen activate protein kinase (MAPK) and the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK) family proteins. These kinases inhibit apoptosis and encourage proliferation. Four human NB cell lines were used to study the effects of Harmine treatment: SK-N-BE(2)-C and KELLY (MYCN amplified) as well as SK-N-AS and SK-N-FI (MYCN single copy). Molecular interaction models of Harmine bound to DYRK family kinases were generated by computational docking using x-ray structures. The anti-cancer properties of Harmine were analyzed by real-time cell viability assays, in a dose-dependent manner, over a 72 hour period. The IC50 values were 169.9, 170.8, and 791.7 μM for SK-N-BE(2)-C, KELLY, and SK-N-FI, respectively, after 72 hours. Apoptosis assays for caspase activation, PARP cleavage, and annexin V induction, were performed using Western blot and flow cytometry. NB cell line exposure to 100 μM Harmine resulted in caspase-3/7 and caspase 9 activation, PARP cleavage, and annexin V-positive stained cells as early as 24 hours after treatment, indicating apoptosis induction. These results led us to investigate the clinical correlations of DYRK family gene expression in NB tumors. The patient results support our hypothesis that Harmine induces NB cell death through a cellular mechanism that involves DYRK family kinases and triggers caspase-mediated apoptosis. Citation Format: Katie L. Uhl, Chad R. Schultz, Dirk Geerts, Andre S. Bachmann. Potential role of DYRK family kinases in harmine-induced apoptosis in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1902.

Abstract 2453: Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA

Abstract N-Myc gene amplification occurs in one quarter of human neuroblastoma tissues, and is a marker for poor patient prognosis. We performed RNA sequencing experiments, and identified 5 transcripts, including RP1NB1, which were most considerably differentially expressed between N-Myc gene amplified and nonamplified human neuroblastoma cell lines. Affymetrix microarray studies revealed that DEPD was one of the few genes considerably downregulated in neuroblastoma cells after RP1NB1 depletion. Chromatin immunoprecipitation assays showed that knocking down RP1NB1 expression reduced histone H3 lysine 4 trimethylation, a marker for active gene transcription, at the DEPD gene promoter. Luciferase assays demonstrated that knocking down RP1NB1 decreased DEPD gene promoter activity. Depletion of RP1NB1 or DEPD with two independent siRNAs or shRNAs significantly reduced ERK protein phosphorylation, N-Myc protein phosphorylation at Serine 62, N-Myc protein stabilization, neuroblastoma cell proliferation and survival. Clonogenic assays showed that knocking down RP1NB1 with doxycycline completely abolished colony formation capacity of neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNAs. Importantly, treatment with doxycycline in mice xenografted with neuroblastoma cells stably transfected with doxycycline-inducible RP1NB1 shRNA led to tumor eradication. In human neuroblastoma tissues from 600 neuroblastoma patients, high levels of RP1NB1 gene expression correlated with DEPD gene expression and poor patient prognosis. In conclusion, this study identifies the novel long noncoding RNA RP1NB1 as an important regulator of N-Myc protein stability and neuroblastoma tumorigenesis. Citation Format: Andrew E. Tee, Pei Y. Liu, Giorgio Milazzo, Kate M. Hannan, Jesper Maag, Nenad Bartonicek, Renhua Song, Chen C. Jiang, Xu D. Zhang, Murray D. Norris, Michelle Haber, Glenn M. Marshall, Jinyan Li, Jo Vandesompele, John S. Mattick, Pieter Mestdagh, Giovanni Perini, Ross D. Hannan, Marcel E. Dinger, Tao Liu. Eradication of neuroblastoma by suppressing the expression of a single noncoding RNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2453.

Plum cultivars grown in Himalayan temperate conditions: physicochemical, antioxidant and antiproliferative activity against three cancer cell lines

Six plum cultivars were evaluated in terms of physicochemical attributes (fruit and stone length, breadth, thickness, weight, fruit moisture content, ash, pH, fruit color, firmness, total soluble solids, total sugars, reducing sugars and titrable acidity), nutraceutical potential i.e., ascorbic acid, β carotene, anthocyanin, total phenol content, total antioxidant activity (DPPH, FRAP) and antiproliferative potential against human breast cancer cell line (MCF7), neuroblastoma (IMR32) and colon cancer cell line (HCT116). The hydroxycinnamates and flavon-3-ols dominated the phenolic profile. Red fleshed plum cultivar Krassivica showed significantly greater antioxidant activity as shown by lowest IC50 value (1.04 mg/mL) for DPPH assay and FRAP value (13.63 µMFe2+/g). Significant inhibition of all the three cancer cells was observed in a dose dependent manner after exposure to plum extracts. For IMR32, MCF7 and HCT116 lowest IC50 value of 67.90, 51.48 and 71.10 µg/mL was observed in Krassivica. Thus, Krassivica can be introduced as a cultivar with good physicochemical characteristics and high neutraceutical potential, making it a preferable choice for fresh use and processing.

Oxidative stress-mediated autophagic cell death participates in the neurotoxic effect on SH-SY5Y cells induced by excessive iodide.

Excessive iodide could induce intellectual damage in children, which has attracted broad attention. To investigate the neurotoxic effect of iodide and its mechanism, a human dopaminergic neuroblastoma cell line (SH-SY5Y) was treated with different concentrations of potassium iodide (KI). The results showed that excessive iodide could decrease cell viability, reduce glutathione (GSH) and superoxide dismutase (SOD), and increase the degree of autophagy (by changing the cellular ultrastructure and raising the autophagy-related mRNA and protein expression of LC3, Beclin1, and p62), which were correlated with the immunofluorescence labeling. Furthermore, treatment with the autophagy inhibitor 3-methyladenine (3MA), antioxidant N-acetylcysteine (NAC) and 30 mM KI for 24 h was conducted in the following research. 3MA significantly decreased autophagy-related mRNA and protein expression and improved cell viability, indicating that excess iodide induced autophagic cell death. In addition, oxidative stress regulated autophagy, reflected by the results that NAC decreased the mRNA and protein expression of LC3, Beclin1, and p62. In summary, autophagic cell death mediated by oxidative stress may participate in excessive iodide-induced SH-SY5Y cell death.

Curcumin Attenuates Hydrogen Peroxide-Induced Cytotoxicity in Human Neuroblastoma SK-N-SH Cells

Objective: Cellular damage induced by oxidative stress has been involved in the development of neurodegeneration. Curcumin, a dietary polyphenol found in the rhizome of Curcuma longa, has been shown, both in vitro and in vivo, to be an effective reactive oxygen scavenging molecule. We investigated an anti-oxidative effect of curcumin against H2O2-induced toxicity in human neuroblastoma cell line SK-N-SH. Methods: The SK-N-SH cells were pre-treated with curcumin 2 hours prior to H2O2 treatment. We measured cell viability, intracellular reactive oxygen species (ROS) levels, expression of apoptotic-related proteins and caspase-3 activity 24 hours post H2O2-induced cytoxicity. Results: Treatment with curcumin at concentrations ranging from 5 to 50 μg/mL was not cytotoxic. Pre-treatment with curcumin at the concentrations of 5 to 50 μg/mL prior to H2O2 exposure caused a significant decrease in intracellular ROS levels and a significant increase in cell viability in a dose-dependent manner. Expression of activated form of caspase-3 and BAX, a pro-apoptotic protein, measured by Western blotting were reduced when the SK-NSH cell line was pre-incubated with curcumin. The curcumin pre-treated cells also exhibited less caspase-3 activity. Conclusion: Curcumin has protective effects against H2O2-induced toxicity in a dose-dependent manner through its anti-apoptotic and anti-oxidative properties in an in vitro H2O2-treated SK-N-SH model.

Arsenic trioxide induces cell cycle arrest and affects Trk receptor expression in human neuroblastoma SK-N-SH cells

BackgroundArsenic trioxide (As2O3), a drug that has been used in China for approximately two thousand years, induces cell death in a variety of cancer cell types, including neuroblastoma (NB). The tyrosine kinase receptor (Trk) family comprises three members, namely TrkA, TrkB and TrkC. Various studies have confirmed that TrkA and TrkC expression is associated with a good prognosis in NB, while TrkB overexpression can lead to tumor cell growth and invasive metastasis. Previous studies have shown that As2O3 can inhibit the growth and proliferation of a human NB cell line and can also affect the N-Myc mRNA expression. It remains unclear whether As2O3 regulates Trks for the purposes of treating NB.MethodsThe aim of the present study was to investigate the effect of As2O3 on Trk expression in NB cell lines and its potential therapeutic efficacy. SK-N-SH cells were grown with increasing doses of As2O3 at different time points. We cultured SK-N-SH cells, which were treated with increasing doses of As2O3 at different time points. Trk expression in the NB samples was quantified by immunohistochemistry, and the cell cycle was analyzed by flow cytometry. TrkA, TrkB and TrkC mRNA expression was evaluated by real-time PCR analysis.ResultsImmunohistochemical and real-time PCR analyses indicated that TrkA and TrkC were over-expressed in NB, and specifically during stages 1, 2 and 4S of the disease progression. TrkB expression was increased in stage 3 and 4 NB. As2O3 significantly arrested SK-N-SH cells in the G2/M phase. In addition, TrkA, TrkB and TrkC expression levels were significantly upregulated by higher concentrations of As2O3 treatment, notably in the 48-h treatment period. Our findings suggested that to achieve the maximum effect and appropriate regulation of Trk expression in NB stages 1, 2 and 4S, As2O3 treatment should be at relatively higher concentrations for longer delivery times;however, for NB stages 3 and 4, an appropriate concentration and infusion time for As2O3 must be carefully determined.ConclusionThe present findings suggested that As2O3 induced Trk expression in SK-N-SH cells to varying degrees and may be a promising adjuvant to current treatments for NB due to its apoptotic effects.