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

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Gang Chen ◽  
Weicheng Chen ◽  
Ming Ye ◽  
Weiqiang Tan ◽  
Bing Jia
Keyword(s):  
Cell Proliferation ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Cell Counting ◽  
Protein Levels ◽  
Proliferation And Apoptosis ◽  
Novel Biomarkers ◽  
Neuroblastoma Cell Lines

Abstract 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.

scholarly journals FOXR2 Stabilizes MYCN Protein and Identifies Non–MYCN-Amplified Neuroblastoma Patients With Unfavorable Outcome

2021 ◽  
pp. JCO.20.02540
Author(s):  
Felix Schmitt-Hoffner ◽  
Sjoerd van Rijn ◽  
Umut H. Toprak ◽  
Monika Mauermann ◽  
Felix Rosemann ◽  
...  
Keyword(s):  
Cell Lines ◽  
Tumor Regression ◽  
Neuroblastoma Cell ◽  
Unfavorable Outcome ◽  
Common Mechanism ◽  
Mycn Amplification ◽  
Data Sets ◽  
Alternative Mechanism ◽  
Protein Levels ◽  
Neuroblastoma Cell Lines

PURPOSE Clinical outcomes of patients with neuroblastoma range from spontaneous tumor regression to fatality. Hence, understanding the mechanisms that cause tumor progression is crucial for the treatment of patients. In this study, we show that FOXR2 activation identifies a subset of neuroblastoma tumors with unfavorable outcome and we investigate the mechanism how FOXR2 relates to poor outcome in patients. MATERIALS AND METHODS We analyzed three independent transcriptional data sets of in total 1030 primary neuroblastomas with full clinical annotation. We performed immunoprecipitation for FOXR2 and MYCN and silenced FOXR2 expression in two neuroblastoma cell lines to examine the effect on cellular processes, transcriptome, and MYCN protein levels. Tumor samples were analyzed for protein levels of FOXR2 and MYCN. RESULTS In three combined neuroblastoma data sets, 9% of tumors show expression of FOXR2 but have low levels of MYCN mRNA. FOXR2 expression identifies a group of patients with unfavorable outcome, showing 10-year overall survival rates of 53%-59%, and proves to be an independent prognostic factor compared with established risk factors. Transcriptionally, FOXR2-expressing tumors are very similar to MYCN-amplified tumors, suggesting that they might share a common mechanism of tumor initiation. FOXR2 knockdown in FOXR2-expressing neuroblastoma cell lines resulted in cell cycle arrest, reduced cell growth, cell death, and reduced MYCN protein levels, all indicating that FOXR2 is essential for these tumors. Finally, we show that FOXR2 binds and stabilizes MYCN protein and MYCN protein levels are highly increased in FOXR2-expressing tumors, in several cases comparable with MYCN-amplified samples. CONCLUSION The stabilization of MYCN by FOXR2 represents an alternative mechanism to MYCN amplification to increase MYCN protein levels. As such, FOXR2 expression identifies another subset of neuroblastoma patients with unfavorable clinical outcome.

scholarly journals Insulin-like Growth Factor-1 Receptor (IGF-1R) As a Potential Therapeutic Target in Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4828-4828
Author(s):  
Xiangxiang Zhou ◽  
Lingyun Geng ◽  
Xinyu Li ◽  
Peipei Li ◽  
Kang Lu ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Caspase 3 ◽  
Cell Lymphoma ◽  
Annexin V ◽  
B Cell Lymphoma ◽  
Control Group ◽  
Protein Levels ◽  
Proliferation And Apoptosis ◽  
Dose Dependent Decrease

Abstract Introduction : The receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) is dysregulated in various tumor entities and hematological malignancies including chronic lymphocytic leukemia and mantle cell lymphoma. The implication of IGF1R in the development and progression of cancer has led to its current evaluation in clinical trials as a potential therapeutic target for solid tumors. However, its functional significance in diffuse large B-cell lymphoma (DLBCL) remains poorly characterized. We hypothesized that IGF1R plays a key role in the pathogenesis and progression of DLBCL. In this present study, we evaluated the expression and function of IGF1R in both B cell lines and DLBCL tissues, as well as assessed the proliferation and apoptosis of DLBCL cells when treated with IGF-1R inhibitor, AG1024. Methods : Expression of IGF1R in B-cell lymphoma cell lines (LY1, LY8, Mino, Jeko-1, and SP53) was evaluated by Western blotting. Peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers with informed consents. Blood samples and araffin-embedded tissues from 30 initial-diagnosed DLBCL patients prior to therapeutic interventions as a study group, and from 15 patients with reactive hyperplasia lymphnode as a control group were collected with informed consents. Immunohistochemisty (IHC) was conducted to assess the expression of IGF-1R in lymphoma tissues. Correlations between IGF1R expression and the clinical characteristics of DLBCL patients were further analyzed. DLBCL cell lines (LY1 and LY8) were treated with an IGF1R specific small molecular inhibitor, AG1024, cell proliferation was analyzed by cell counting kit (CCK-8). Effects of inhibitor or stimulator on the apoptosis of LY1 and LY8 cells were assessed by Annexin-V/PI and Annexin-V/7AAD, respectively. Expression of apoptosis-related protein, including Caspase-3 and Mcl-1, was evaluated by western blotting. Protein levels of downstream targets of IGF-1 signaling were also detected. Results : Significantly upregulation of both phoaphprylated and total IGF1R protein levels were found in B-cell lymphoma cells (LY1, LY8, Mino, Jeko-1 and SP53) (Fig 1.A). IHC was conducted and revealed significantly enhancement of IGF1R expression in DLBCL patients (Fig 1.B). Among the included DLBCL patients and control group with inreactive hyperplastic lymphadenitis, the positive rate of IGF1R was 90% and 20%, respectively. We then investigated the function of IGF1R inhibitors on the proliferation and apoptosis of DLBCL cells. LY8 cells were treated with different doses of AG1024 at 24-96 hours. Cell proliferation was inhibited by 60% when treated with AG1024 at the concentration of 15µM for 72-hours (Fig 1.C). Culture of LY1 and LY8 cells in the presence of 10µM and 15µM AG1024 concentration for 24-hours resulted in 13% (p<0.05) and 33% (p<0.001) cell apoptosis, respectively (Fig 1.D). Inhibition of IGF1R by AG1024 also resulted in induction of cleaved-Caspase-3, as well as reduction of Mcl-1(Fig 1.E-F). In order to investigate the mechanisms involved in the dysregultaion of IGF1R in DLBCL, LY8 cells were treated with 5 to 15 µM AG1024, the results revealed that AG1024 caused a dose-dependent decrease in the levels of phosphorylated IGF1R, AKT and ERK (Fig 1.G). Treatment of LY8 cells with recombinant human IGF-1 led to enhanced phosporylation levels of IGF1R, AKT and ERK (Fig 1.H). Conclusion s: Our investigation observed that expression levels of IGF-1R were up-regulated in both B-cell lymphoma cells and DLBCL tissues. DLBCL cells treated with IGF-1R inhibitor, AG1024, revealed reduced proliferation and increased apoptosis rate. In addition, induction of cleaved-Caspase-3 was also found in LY1 treated with AG1024. AG1024 caused a dose-dependent decrease in the phosphorylation levels of IGF1R, AKT and ERK. This study suggests that IGF1R could be a potential molecular target for the treatment of DLBCL. The IGF-1R inhibitor is a promising therapeutic approach for DLBCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

2021 ◽  
Vol 3 (4) ◽  
pp. 12-24
Author(s):  
Mabao YUAN ◽  
Hanjiao HANG ◽  
Lubin YAN ◽  
Xuanjie HUANG ◽  
Ziyang SANG ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Luciferase Reporter ◽  
Ubiquitin Protein Ligase ◽  
Neuroblastoma Cell Lines ◽  
Dual Luciferase Reporter Assay

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

scholarly journals Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest

2019 ◽  
Vol 28 (1) ◽  
pp. 126-133 ◽  
Author(s):  
Thatyanne Gradowski do Nascimento ◽  
Priscilla Santos Vieira ◽  
Sheron Campos Cogo ◽  
Marcela Ferreira Dias-Netipanyj ◽  
Nilton de França Junior ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Fluorescent Microscopy ◽  
Annexin V ◽  
Hard Tick ◽  
Antitumor Properties ◽  
Vitro Effect ◽  
Neuroblastoma Cell Lines

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Long noncoding RNA SNHG14 regulates ox-LDL-induced atherosclerosis cell proliferation and apoptosis by targeting miR-186-5p/WIPF2 axis

2020 ◽  
Vol 40 (1) ◽  
pp. 47-59
Author(s):  
Z Tao ◽  
Z Cao ◽  
X Wang ◽  
D Pan ◽  
Q Jia
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Noncoding Rna ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Interacting Protein ◽  
Downstream Target ◽  
Proliferation And Apoptosis

To investigate the role of small nucleolus RNA host gene 14 (SNHG14) in the progression of atherosclerosis (AS), bioinformatics analysis, and other relevant experiments (cell counting kit-8, flow cytometry, quantitative real-time polymerase chain reaction, luciferase reporter, RNA immunoprecipitation, RNA pull-down, and western blot assays) were done. The current study revealed that SNHG14 level was high in the serum of AS patients and oxidized low-density lipoprotein (ox-LDL)-induced AS cell lines. Besides, we found that SNHG14 accelerated cell proliferation while inhibited cell apoptosis in ox-LDL-induced AS cell lines. Next, SNHG14 was confirmed to be a sponge for miR-186-5p in AS cells, and it was validated that SNHG14 regulated AS cell proliferation and apoptosis by sponging miR-186-5p. Moreover, we uncovered that WAS-interacting protein family member 2 (WIPF2) was a downstream target of miR-186-5p in AS cells. Finally, it was demonstrated that miR-186-5p modulated AS cell proliferation and apoptosis via targeting WIPF2. To conclude, our research disclosed that SNHG14 affected ox-LDL-induced AS cell proliferation and apoptosis through miR-186-5p/WIPF2 axis, which may provide a theoretical basis for the treatment and diagnosis of AS.

scholarly journals In Vitro Assessment of the Role of p53 on Chemotherapy Treatments in Neuroblastoma Cell Lines

2021 ◽  
Vol 14 (11) ◽  
pp. 1184
Author(s):  
Idoia Blanco-Luquin ◽  
Paula Lázcoz ◽  
Jon Celay ◽  
Javier S. Castresana ◽  
Ignacio J. Encío
Keyword(s):  
Retinoic Acid ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Wild Type ◽  
Expression Levels ◽  
Homozygous Deletions ◽  
Neuroblastoma Cell Lines ◽  
Mdm2 Amplification

Neuroblastoma is the most frequent malignant extracranial solid tumor of infancy. The overall objective of this work consists of determining the presence of alterations in the p53/MDM2/p14ARF signaling pathway in neuroblastoma cell lines and deciphering their possible relationship with resistance to known antineoplastic drugs and to differentiation agents. Firstly, we characterized 10 neuroblastoma cell lines for alterations at the p53/MDM2/p14ARF signaling pathway by analysis of TP53 point mutations, MYCN and MDM2 amplification, and p14ARF methylation, homozygous deletions, and expression. Secondly, we chose SK-N-FI (mutated at TP53) and SK-N-Be(2) (wild-type TP53) cell lines, treated them with chemotherapeutic agents (doxorubicin, etoposide, cisplatin, and melphalan) and with two isomers of retinoic acid (RA): (9-cis and all-trans). Finally, we analyzed the distribution of the cell cycle, the induction of apoptosis, and the expression levels of p53, p21, and Bcl-2 in those two cell lines. P14ARF did not present promoter methylation, homozygous deletions, and protein expression in any of the 10 neuroblastoma cell lines. One TP53 point mutation was detected in the SK-N-FI cell line. MYCN amplification was frequent, while most cell lines did not present MDM2 amplification. Treatment of SK-N-FI and SK-N-Be(2) cells with doxorubicin, etoposide, cisplatin, and melphalan increased apoptosis and blocked the cycle in G2/M, while retinoic acid isomers induced apoptosis and decreased the percentage of cells in S phase in TP53 mutated SK-N-FI cells, but not in TP53 wild-type SK-N-Be(2) cells. Treatment with cisplatin, melphalan, or 9-cis RA decreased p53 expression levels in SK-N-FI cells but not in SK-N-Be (2). The expression of p21 was not modified in either of the two cell lines. Bcl-2 levels were reduced only in SK-N-FI cells after treatment with cisplatin. However, treatments with doxorubicin, etoposide, or 9-cis-RA did not modify the levels of this protein in either of the two cell lines. In conclusion, TP53 mutated SK-N-FI cells respond better to the retinoic isomers than TP53 wild-type SK-N-Be(2) cells. Although these are in vitro results, it seems that deciphering the molecular alterations of the p53/MDM2/p14ARF signaling pathway prior to treating patients of neuroblastoma might be useful for standardizing therapies with the aim of improving survival.

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