Apoptosis In Neuroblastoma Cell Lines Research Articles

A direct comparison of selective BH3-mimetics reveals BCL-XL, BCL-2 and MCL-1 as promising therapeutic targets in neuroblastoma

BackgroundDespite advances in the treatment of neuroblastoma, patients with high-risk disease still have dismal survival prognosis. Neuroblastoma cells display elevated expression of the antiapoptotic BCL-2 proteins, suggesting that BH3-mimetics may be a promising treatment option. Here, we investigated the role of BCL-2, BCL-XL and MCL-1 in neuroblastoma.MethodsA panel of neuroblastoma cell lines and primary patient-derived cells were exposed to BH3-mimetics targeting BCL-2 (ABT-199), BCL-XL (A1331852) or MCL-1 (S63845). In addition, protein expression and interaction patterns were analysed using Western blotting and immunoprecipitation.ResultsAll tested BH3-mimetics were able to induce apoptosis in neuroblastoma cell lines, indicating that not only BCL-2 but also BCL-XL and MCL-1 may be promising therapeutic targets. Primary patient-derived cells displayed highest sensitivity to A1331852, highlighting the important role of BCL-XL in neuroblastoma. Further analysis into the molecular mechanisms of apoptosis revealed that A1331852 and S63845 displaced proapoptotic proteins like BIM and BAK from their antiapoptotic targets, subsequently leading to the activation of BAX and BAK and caspase-dependent apoptosis.ConclusionsBy using selective BH3-mimetics, this study demonstrates that BCL-2, BCL-XL, and MCL-1 are all relevant therapeutic targets in neuroblastoma. A1331852 and S63845 induce rapid apoptosis that is initiated following a displacement of BAK from BCL-XL or MCL-1, respectively.

MiR-490-5p functions as tumor suppressor in childhood neuroblastoma by targeting MYEOV.

Neuroblastoma (NB) is the most common extracranial solid tumor derived from neural crest in children. Recently, the role of miRNA has been studied extensively in the development of NB. Here, we investigated the clinical significance of microRNA-490-5p (miR-490-5p) in NB. A total of 72 pairs of NB tumor tissues and matched adjacent normal nerve tissues were collected from NB patients. The expression of miR-490-5p was significantly down-regulated in NB tissues and cell lines using quantitative real-time PCR. Using Pearson Chi-square test and Kaplan-Meier analysis, we found that significantly decreased miR-490-5p levels were correlated with INSS stage, lymph-node metastasis, and poor survival prognosis in NB patients. MiR-490-5p overexpression significantly suppressed cell proliferation migration, invasion, and induced cell cycle G0/G1 arrest and cell apoptosis in NB cell lines (SH-SY5Y and SK-N-SH) using CCK-8, flow cytometry, and transwell assays. Mechanistically, MYEOV was confirmed as a target gene of miR-490-5p by luciferase reporter assay. Furthermore, MYEOV knockdown imitated, while overexpression rescued the changes in the biological features of miR-490-5p on NB cells. Our results demonstrated for the first time that miR-490-5p functions as a tumor suppressor in NB by targeting MYEOV, which might provide novel approaches for the treatment of NB.

TRIM59 knockdown inhibits cell proliferation by down-regulating the Wnt/β-catenin signaling pathway in neuroblastoma.

Neuroblastoma is the most common tumor in children, with a very poor prognosis. It is urgent to identify novel biomarkers to treat neuroblastoma, together with surgery, chemotherapy, and radiation. Human tripartite motif 59 (TRIM59), a member of the TRIM family, has been reported to participate in several human tumors. However, the exact role of TRIM59 in neuroblastoma is unknown. In the present study, real-time PCR and Western blot were used to measure mRNA and protein levels of TRIM59 in four neuroblastoma cell lines and in neuroblastoma tissues. Lentiviruses targeting TRIM59 were used to up/down-regulate TRIM59 expression levels. Cell Counting Kit-8 and Annexin-V/PI were used to analyze cell proliferation and apoptosis in neuroblastoma cell lines. Our data showed that TRIM59 knockdown inhibits cell proliferation while inducing apoptosis in SH-SY5Y and SK-N-SH neuroblastoma cell lines. TRIM59 knockdown up-regulated expression of Bax and Bim and down-regulated levels of Survivin, β-catenin, and c-myc. Interestingly, the inhibition of cell proliferation caused by TRIM59 knockdown could be blocked by LiCl, which is an agonist of Wnt/β-catenin signaling pathway. In contrast, TRIM59 overexpression could increase cell proliferation, up-regulate Survivin, β-catenin and c-myc, down-regulate Bax and Bim, and these effects could be blocked by XAV939, which is an inhibitor of Wnt/β-catenin signaling pathway. In addition, TRIM59 was up-regulated and positively related with β-catenin in neuroblastoma tissues. In conclusion, TRIM59 was up-regulated in neuroblastoma, and TRIM59 knockdown inhibited cell proliferation by down-regulating the Wnt/β-catenin signaling pathway in neuroblastoma.

Silica nanoparticles induce conformational changes of tau protein and oxidative stress and apoptosis in neuroblastoma cell line

The adverse effects of SiO2 NPs on the biological systems like nervous system have not been well explored. This study aimed to evaluate the toxicity of SiO2 NPs on the nervous system in vitro. Therefore, human tau protein and neuroblastoma cell line (SH-SY5Y) were used as targets. In this study we examined the side effects of SiO2 NPs on tau protein structure using several techniques including CD, ANS fluorescence, UV–vis (360 nm), Congo red absorbance, TEM, and molecular dynamic. Also, the cytotoxicity effects of SiO2 NPs against SH-SY5Y cell line were evaluated using MTT, ROS and apoptotic assays. Spectroscopic and molecular dynamic investigations indicated that natively unfolded structure of tau in the presence of SiO2 NPs experienced a partially folded and amorphous aggregated structure. Cellular assay demonstrated that SiO2 NPs exerted cytotoxic effect on SH-SY5Y cells through ROS accumulation and induction of apoptosis. Overall, these findings proved that SiO2 NPs could induce adverse effects on tau structure and SH-SY5Y cell integrity. Moreover, further studies are required to elucidate the molecular mechanism of SiO2 NPs-induced side effects in vivo.

Inhibition of neuroblastoma proliferation by PF-3758309, a small-molecule inhibitor that targets p21-activated kinase 4

Neuroblastoma is the most common extracranial solid childhood tumor. Despite the availability of advanced multimodal therapy, high-risk patients still have low survival rates. p21-activated kinase 4 (PAK4) has been shown to regulate many cellular processes in cancer cells, including migration, polarization and proliferation. However, the role of PAK4 in neuroblastoma remains unclear. In the present study, we demonstrated that PAK4 was overexpressed in neuroblastoma tissues and was correlated with tumor malignance and prognosis. To investigate the function of PAK4 in neuroblastoma, we used a small-molecule inhibitor that targets PAK4, that is, PF-3758309. Our results showed that PF-3758309 significantly induced cell cycle arrest at the G1 phase and apoptosis in neuroblastoma cell lines. Meanwhile, the inhibition of PAK4 by PF-3758309 increased the expression of CDKN1A, BAD and BAK1 and decreased the expression of Bcl-2 and Bax. In addition, we screened the target genes of PAK4 by PCR array and found that 23 genes were upregulated (including TP53I3, TBX3, EEF1A2, CDKN1A, IFNB1 and MAPK8IP2) and 20 genes were downregulated (including TNFSF8, Bcl2-A1, Bcl2L1, SOCS3, BIRC3 and NFKB1) after PAK4 inhibition by PF-3758309. Moreover, PAK4 was found to regulate the cell cycle and apoptosis via the ERK signaling pathway. In conclusion, the present study demonstrated, for the first time, the expression and function of PAK4 in neuroblastomas and the inhibitory effect of PF-3758309, which deserves further investigation as an alternative strategy for neuroblastoma treatment.

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB.

Targeting tachykinin receptors in neuroblastoma.

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

NSC-87877 inhibits DUSP26 function in neuroblastoma resulting in p53-mediated apoptosis.

Dual specificity protein phosphatase 26 (DUSP26) is overexpressed in high-risk neuroblastoma (NB) and contributes to chemoresistance by inhibiting p53 function. In vitro, DUSP26 has also been shown to effectively inhibit p38 MAP kinase. We hypothesize that inhibiting DUSP26 will result in decreased NB cell growth in a p53 and/or p38-mediated manner. NSC-87877 (8-hydroxy-7-[(6-sulfo-2-naphthyl)azo]-5-quinolinesulfonic acid), a novel DUSP26 small molecule inhibitor, shows effective growth inhibition and induction of apoptosis in NB cell lines. NB cell lines treated with small hairpin RNA (shRNA) targeting DUSP26 also exhibit a proliferation defect both in vitro and in vivo. Treatment of NB cell lines with NSC-87877 results in increased p53 phosphorylation (Ser37 and Ser46) and activation, increased activation of downstream p38 effector proteins (heat shock protein 27 (HSP27) and MAP kinase-activated protein kinase 2 (MAPKAPK2)) and poly ADP ribose polymerase/caspase-3 cleavage. The cytotoxicity resulting from DUSP26 inhibition is partially reversed by knocking down p53 expression with shRNA and also by inhibiting p38 activity with SB203580 (4-[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine). In an intrarenal mouse model of NB, NSC-87877 treatment results in decreased tumor growth and increased p53 and p38 activity. Together, these results suggest that DUSP26 inhibition with NSC-87877 is an effective strategy to induce NB cell cytotoxicity in vitro and in vivo through activation of the p53 and p38 mitogen-activated protein kinase (MAPK) tumor-suppressor pathways.

XAV939, a tankyrase 1 inhibitior, promotes cell apoptosis in neuroblastoma cell lines by inhibiting Wnt/β-catenin signaling pathway

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in childhood. The present treatment including surgery, chemotherapy and radiation, which have only 40% long-term cure rates, and usually cause tumor recurrence. Thus, looking for new effective and less toxic therapies has important significance. XAV939 is a small molecule inhibitor of tankyrase 1(TNKS1). The objective of this study is to investigate the effect of XAV939 on the proliferation and apoptosis of NB cell lines, and the related mechanism.MethodsIn the present study, we used both XAV939 treatment and RNAi method to demonstrate that TNKS1 inhibition may be a potential mechanism to cure NB. MTT method was used for determining the cell viability and the appropriate concerntration for follow-up assays. The colony formation assay, Annexin V staining and cell cycle analysis were used for detecting colony forming ability, cell apoptosis and the percentage of different cell cycle. The Western blot was used for detecting the expression of key proteins of Wnt/ beta-catenin (Wnt/β-catenin) signaling pathway.ResultsThe results showed that TNKS1 inhibition decreased the viability of SH-SY5Y, SK-N-SH and IMR-32 cells, induced apoptosis in SH-SY5Y as well as SK-N-SH cells, and led to the accumulation of NB cells in the S and G2/M phase of the cell cycle. Moreover, we demonstrated TNKS1 inhibition may in part blocked Wnt/β-catenin signaling and reduced the expression of anti-apoptosis protein. Finally, we also demonstrated that TNKS1 inhibition decreased colony formation in vitro.ConclusionsThese findings suggested that TNKS1 may be a potential molecule target for the treatment of NB.

Emerging treatment options for the treatment of neuroblastoma: potential role of perifosine

Achieving a cure for high-risk neuroblastoma, the most common extracranial solid tumor in children, remains a formidable task despite the recent addition of antibody-mediated anti-GD2 immunotherapy to established multimodality therapy. The PI3K/Akt pathway is a pivotal signaling pathway utilized by a plethora of receptor tyrosine kinases that contribute to the aggressive phenotype of high-risk neuroblastoma. Akt is aberrantly activated in high-risk neuroblastoma and is therefore an attractive therapeutic target. Perifosine is the best-characterized Akt inhibitor in preclinical studies and in clinical trials in adults, although safety in children is not yet confirmed. It is a synthetic third-generation alkylphospholipid with good oral bioavailability and modest side effects. Perifosine targets the lipid-binding PH domain of Akt and inhibits the translocation of Akt to the cell membrane, an essential step for Akt activation. It decreases Akt phosphorylation and increases caspase-dependent apoptosis in neuroblastoma cell lines, inhibits growth of neuroblastoma xenografts, and overcomes RTK/ligand-mediated chemoresistance. It is currently being studied in two Phase I clinical trials in children with recurrent or refractory solid tumors including neuroblastoma. In the single agent trial (ClinicalTrials.gov identifier NCT00776867), maximum tolerated dose has not yet been reached and pharmacokinetic data has been accrued. In the second study (ClinicalTrials.gov identifier NCT01049841), patients are treated with a combination of perifosine and the mTOR-inhibitor temsirolimus based on preclinical data showing synergy of the two agents, and the premise that direct Akt inhibition may overcome Akt activation secondary to mTOR inhibition. Based on results from adult trials, it is unlikely that perifosine alone will produce dramatic therapeutic effects against high-risk neuroblastoma. However, given the recent encouraging early-phase combination therapy results in adults with multiple myeloma and colorectal carcinoma, rational perifosine-containing combination regimens hold promise for neuroblastoma therapy. These will be explored after safety in children is established in Phase I studies.

The impact of retinoic acid treatment on the sensitivity of neuroblastoma cells to fenretinide

Despite the successful introduction of 13-cis retinoic acid (13cisRA) therapy for the treatment of neuroblastoma, approximately 50% patients do not respond or experience relapse. A retinoid analogue, fenretinide [N-(4-hydroxyphenyl) retinamide; 4-HPR] can induce apoptosis in neuroblastoma cell lines and could have clinical use after therapy with 13cisRA. However, there are important questions concerning potential retinoid drug interactions which need to be addressed. The aim of this study was to investigate the influence of retinoic acid pre-treatment on fenretinide-induced apoptosis and fenretinide metabolism in neuroblastoma cell lines. Apoptosis was measured by flow cytometry of propidium iodide-stained neuroblastoma cells and a live-cell imaging assay. Intracellular fenretinide metabolism was determined by HPLC analysis. Pre-treatment of neuroblastoma cell lines with retinoic acid (RA) resulted in a significant decrease in the apoptotic response to fenretinide in three of the four lines tested. Comparison between responsive and non-responsive cell lines suggested that RA sensitivity was required to promote fenretinide resistance, and that this was mediated by up-regulation of Bcl-2 and the inhibition of pro-apoptotic fenretinide signalling pathways. Induction of the oxidative metabolism of fenretinide after RA pre-treatment did not significantly impact on intracellular parent drug levels and is unlikely to explain the decreased apoptotic response observed. The interaction between RA and fenretinide could have important implications for the scheduling of fenretinide in therapeutic protocols for neuroblastoma.

Stable knockdown of MYCN by lentivirus-based RNAi inhibits human neuroblastoma cells growth in vitro and in vivo

Neuroblastoma is the most common childhood solid tumor, yet current treatment approaches have not been able to effectively control this cancer. Amplification and overexpression of MYCN have been shown to be closely related with high risk and poor prognosis in neuroblastoma. This suggests that MYCN is an important target for the antitumor therapy. Recently, vector-based RNA interference (RNAi) systems have been successfully used to eliminate gene expression, but knockdown of MYCN by vector-based RNAi as a therapeutic model for neuroblastoma has not been fully established.In this study, we used a lentivirus vector-based RNAi approach which expresses short hairpin RNA (shRNA) to knockdown MYCN in neuroblastoma cell lines IMR-32 and LAN-1. Western blotting analysis showed that expressions of MYCN were efficiently downregulated after infection with MYCN shRNA expression vector. The stable suppression of MYCN expression induced differentiation and apoptosis in neuroblastoma cell lines. Furthermore, we demonstrated that these changes were associated with caspase-3 activation, p27 upregulation as well as Bcl-2 and MDM2 downregulation. Finally, we demonstrated that downregulation of MYCN expression significantly reduced colony formation in vitro and tumor growth in nude mice.Our data indicate that lentivirus vector-mediated silencing of MYCN in neuroblastoma cells could efficiently and significantly inhibit tumor growth both in vitro and in vivo. Therefore we demonstrate the therapeutic potential of lentivirus-delivered shRNA as a novel approach for treatment of neuroblastoma and other malignant tumors with MYCN overexpression.

Abstract 4341: Bortezomib primes neuroblastoma cells for TRAIL- triggered apoptosis by linking the death receptor to the mitochondrial pathway

Abstract In contrast to remarkable improvements in the survival and cure rates that have been encountered for many childhood malignancies, the prognosis of children with advanced stage neuroblastoma remains poor. Searching for novel strategies to modulate apoptosis in neuroblastoma we investigated the potential of the proteasome inhibitor Bortezomib to modulate TRAIL-induced apoptosis in neuroblastoma cell lines, primary neuroblastoma cultures and in an in vivo model of neuroblastoma. Here, we provide first evidence that Bortezomib at subtoxic concentrations synergistically cooperates with TRAIL to induce apoptosis (combination index 0.5). In addition, Bortezomib acts in concert with TRAIL to reduce colony formation of neuroblastoma cells, demonstrating an effect also on longterm survival. Mechanistic studies reveal that Bortezomib profoundly enhances TRAIL-induced cleavage of Bid into tBid, accumulation of tBid in the cytosol and its insertion into mitochondrial membranes, pointing to a concerted effect on Bid cleavage (TRAIL) and stabilization of tBid (Bortezomib), which links the death receptor to the mitochondrial pathway. Additionally, Bortezomib increases expression of p53 and Noxa. All these changes lead to increased activation of Bax and Bak, loss of the mitochondrial membrane potential, cytochrome c release, caspase activation and caspase-dependent apoptosis upon treatment with Bortezomib and TRAIL. Knockdown of either Bid, Noxa or p53 significantly delays the kinetic of Bortezomib- and TRAIL-induced apoptosis, whereas it does not confer longterm protection. By comparison, overexpression of Bcl-2, which simultaneously antagonizes tBid, Noxa and p53, significantly inhibits Bortezomib- and TRAIL-induced apoptosis and even rescues clonogenic survival. Of note, Bortezomib and TRAIL act in concert to trigger apoptosis in several patients’ derived primary neuroblastoma cultures, underscoring the clinical relevance. Importantly, Bortezomib cooperates with TRAIL to suppress tumor growth in an in vivo model of neuroblastoma. In conclusion, these findings demonstrate for the first time that Bortezomib at subtoxic concentrations represents a promising new approach to prime neuroblastoma cells towards TRAIL-induced apoptosis by linking the death receptor to the mitochondrial pathway. Thus, this study provides the rational for future (pre)clinical evaluation of the combination strategy with Bortezomib and TRAIL in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4341. doi:10.1158/1538-7445.AM2011-4341

All-trans retinoic acid downregulates ALK in neuroblastoma cell lines and induces apoptosis in neuroblastoma cell lines with activated ALK

Recently, gene amplification and gain-of-function mutations of ALK have been found in some neuroblastoma cell lines and clinical tumor samples. We have previously reported that knockdown of ALK by RNAi induced apoptosis in neuroblastoma cells with gene amplification of ALK. We report that all-trans retinoic acid (ATRA) downregulates ALK in neuroblastoma cell lines. Downregulation of ALK protein by ATRA was accompanied by apoptosis in neuroblastoma cells with gene amplification or gain-of-function mutation of ALK but not in neuroblastoma cells without these genetic alterations. These results suggest that ALK downregulation by ATRA might lead to apoptosis in neuroblastoma cells with activated ALK.

Abstract 666: Regulation of IGF-1 signaling by lysosomal cathepsins in neuroblastoma

Abstract Neuroblastoma is the most common extracranial solid tumor of childhood. Despite intensive combination therapies, the survival rate for children diagnosed with high-risk neuroblastoma remains below 40%, with surviving children suffering from severe side-effects of aggressive surgery, cytotoxic chemotherapy and radiation. We show that specific inhibition of a group of lysosomal proteases results in accumulation of autophagic vacuoles and eventual apoptosis of neuroblastoma cell lines. Cell death is not caused in breast and prostate cancer cell lines or mouse primary fibroblasts, indicating specificity of the apoptotic response in neuroblastoma. In this study we have examined mechanisms by which inhibition of these proteases can lead to the specific death of these tumors. IGF-1 signaling is essential for cellular proliferation, survival, and apoptotic resistance. We found that in neuroblastoma cells IGF-1 signaling is impaired by inhibition of cathepsins B and L. Western blotting shows that lower molecular weight fragments of the IGF-1 receptor beta subunit accumulate in inhibitor-treated cells, indicating that lysosomal proteases are essential for the complete the degradation of the IGF-1 receptor. Phosphorylation and activation of full-length IGF-1 receptor was not modified by inhibitor treatment, but phosphorylation and activation of MAPK and Akt (downstream effectors of IGF-1 receptor signaling) was greatly diminished. Fractionation of cellular lysates showed that the IGF-1R β fragments accumulate in compartments that contain a cleaved form of LC-3, a marker of autophagic vacuoles. Intermediates of the IGF-1 signaling cascade were also sequestered within these compartments, and significantly depleted in the cytoplasm. We conclude that inhibition of lysosomal proteases results in sequestration of critical components of the cell signaling transduction pathway, causing cell death by uncoupling of cell surface and endosomal signaling from intracellular effector signals. 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 666.

73. Triptolide Therapy for Neuroblastoma Decreases Cell Viability in Vitro and Inhibits Tumor Growth in Vivo

Neuroblastoma is the most common pediatric extracranial tumor, and advanced-stage cases are highly resistant to conventional chemotherapy. Hsp-70 is overexpressed in several human malignancies, and its inhibition has been shown to kill cancer cells. We have recently shown that triptolide, an inhibitor of Hsp-70, induces apoptotic cell death of pancreatic tumors. Our primary objective was to assess the effectiveness of triptolide therapy in treatment of neuroblastoma, in vitro and in vivo. Additional aims were to assess the effect of triptolide treatment on levels of Hsp 70 and markers of apoptosis in neuroblastoma cell lines.

Selenium protects the hypoxia induced apoptosis in neuroblastoma cells through upregulation of Bcl-2

Selenium (Se) is an essential micronutrient as well as a toxic trace element in animal and human nutrition. The effects of Se in the immune system and some diseases are well documented. The objective of the present study was to examine the role of Se in reducing the hypoxia induced apoptosis in neuroblastoma cell line. Hypoxia showed an enhanced cytotoxicity, increased free radical production and apoptosis ( p < 0.001) which was measured in terms of DNA break down by comet assay. Hypoxia has decreased reduced Glutathione (GSH) content, Glutathione Reductase (GR), Glutathione peroxidase (GPx) and Superoxide Dismutase (SOD) activities as compared to control cells. During hypoxic condition the expression of cytochrome C, pro and active caspase-3 levels were enhanced significantly followed by nonsignificant upregulation of Bcl-2. But, the Se supplementation inhibited the cytotoxicity, free radical generation and stabilized the HIF-1α accumulation in cells under hypoxia. The GSH content, GR, GPx and SOD activities increased significantly in Se-treated hypoxic cells, as compared to control. Further there was an appreciable inhibition of apoptosis by upregulation of Bcl-2 proteins, in the presence of Se under hypoxia. Selenium supplementation to cells significantly inhibited the hypoxia induced DNA fragmentation and restored the antioxidant status back to control levels. This study suggests that Se supplementation prevented the cells from hypoxia induced apoptosis by triggering upregulation of Bcl-2 protein and reducing the oxidative stress.

B-006 Preparation of folate ornamentation PEG-liposome containing ceramide-induced apoptosis of neuroblastoma cell lines(Oral Presentation Oncology 1,Better Life for Sick Children, Better Future for Pediatric Surgery,the 45th Annual Meeting of Japanese Society of Pediatric Surgeons)

B-006 Preparation of folate ornamentation PEG-liposome containing ceramide-induced apoptosis of neuroblastoma cell lines(Oral Presentation Oncology 1,Better Life for Sick Children, Better Future for Pediatric Surgery,the 45th Annual Meeting of Japanese Society of Pediatric Surgeons)

Sodium Ascorbate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake

BackgroundNeuroblastoma (NB) is an extra-cranial solid tumour of childhood. In spite of the good clinical response to first-line therapy, complete eradication of NB cells is rarely achieved. Thus, new therapeutic strategies are needed to eradicate surviving NB cells and prevent relapse. Sodium ascorbate has been recently reported to induce apoptosis of B16 melanoma cells through down-regulation of the transferrin receptor, CD71. Since NB and melanoma share the same embryologic neuroectodermal origin, we used different human NB cell lines to assess whether the same findings occurred.ResultsWe could observe dose- and time-dependent induction of apoptosis in all NB cell lines. Sodium ascorbate decreased the expression of CD71 and caused cell death within 24 h. An increase in the global and specific caspase activity took place, as well as an early loss of the mitochondrial transmembrane potential. Moreover, intracellular iron was significantly decreased after exposure to sodium ascorbate. Apoptotic markers were reverted when the cells were pretreated with the iron donor ferric ammonium citrate (FAC), further confirming that iron depletion is responsible for the ascorbate-induced cell death in NB cells.ConclusionSodium ascorbate is highly toxic to neuroblastoma cell lines and the specific mechanism of vitamin C-induced apoptosis is due to a perturbation of intracellular iron levels ensuing TfR-downregulation.

UCN-01 alters phosphorylation of Akt and GSK3beta and induces apoptosis in six independent human neuroblastoma cell lines.

In this study we evaluated UCN-01, a small molecule that inhibits protein kinases by interacting with the ATP-binding site, as a potential anti-cancer agent for neuroblastoma. UCN-01 was effective at inducing apoptosis in six neuroblastoma cell lines with diverse cellular and genetic phenotypes. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assays, detection of active caspase-3 and cleaved poly ADP-ribose polymerase (PARP) confirmed that UCN-01 induced apoptosis. Cell cycle analysis determined that the UCN-01 treated cells accumulated in S phase by 16 h. Unlike vinblastine and docetaxel that increased survivin expression, UCN-01 treatment did not increase X-linked inhibitor of apoptosis protein (XIAP) and survivin levels. Analysis of specific phosphoepitopes on chk1/2, Akt, and GSK3beta following UCN-01 treatment determined that there was no significant change in phospho-chk1/2. However, there was decreased immunoreactivity at Ser473 and Thr308 of Akt and Ser9 of GSK3beta by 4 h indicating that the Akt survival pathway and downstream signalling was compromised. Thus, UCN-01 was effective at inducing apoptosis in neuroblastoma cell lines.