Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells

Horwacik, Irena; Durbas, Małgorzata; Boratyn, Elżbieta; Węgrzyn, Paulina; Rokita, Hanna

doi:10.1016/j.canlet.2013.08.018

Cited by 34 publications

(94 citation statements)

References 44 publications

(84 reference statements)

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“…The hybridoma cell line producing the 14G2a mAb (IgG2a), binding to GD2, was kindly provided by Dr R.A. Reisfeld (Scripps Institute, La Jolla, CA, USA). Cells were cultured as described in detail (17). the 14g2a mab was purified from Fbs (fetal bovine serum) free cell culture supernatant as described in detail (17).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were cultured as described in detail (17). the 14g2a mab was purified from Fbs (fetal bovine serum) free cell culture supernatant as described in detail (17).…”

Section: Methodsmentioning

confidence: 99%

“…The 14G2a mAb (IgG2a) was used to detect presence of GD2 ganglioside on cell clones (WT, Mock1, S2, S4). The PK136 mAb (IgG2a) was used as an isotype control (17). Binding of the antibodies were detected using mouse Ig-specific FITC-conjugated goat F(ab') 2 fragments (cat.…”

Section: Lentiviral Transduction Imr-32 (25x10mentioning

confidence: 99%

“…55526, Cappel, MP Biomedicals, LLC, Warszawa, Poland), a detailed protocol can be seen in ref. 17. Cell cycle analyses were performed using the protocols for adherent cells described in detail (21).…”

Section: Lentiviral Transduction Imr-32 (25x10mentioning

confidence: 99%

“…18, and a specific aurora a inhibitor Mk-5108 increased expression of PHLDA1, when used alone or combined together (17). Incubation of the mAb with IMR-32 neuroblastoma cell line led to cell death with involvement of apoptosis (17,18) and this correlated with a decrease in the Aurora A protein level and its activating phosphorylation, a decrease of the cytoplasmic level of MYCN, and an increase in the p53 level (17). based on our prior findings, we aimed to further study roles of PHLDA1 in our model.…”

Section: Introductionmentioning

confidence: 98%

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Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Durbas

Horwacik

Boratyn

et al. 2016

International Journal of Oncology

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Abstract.We have recently shown that mRNA and protein of PHLDA1 (pleckstrin-homology-like domain family A, member 1) were by far the most upregulated molecules upon treatment of IMR-32 cells with the anti-GD2 ganglioside monoclonal antibody 14G2a. Hence, we decided to study functions of PHLDA1 using human neuroblastoma IMR-32 cells as a model. Here, we show that constitutive expression of mRNA and protein of the PHLDA1 gene in IMR-32 cells was inversely correlated with transcript of the AURKA gene and Aurora A oncoprotein. Next, we silenced PHLDA1 expression in IMR-32 cells using an shRNA interference method. We report that IMR-32 cells with stable downregulation of PHLDA1 showed enhanced cellular ATP levels and an increase in mitochondrial membrane potential, as compared to control and non-transduced cells. We demonstrated that downregulation of PHLDA1 leads to a significant increase in expression of Aurora A and TRKB that are markers of poor prognosis in neuroblastoma. Also, we measured an increase in Aurora A and Akt kinases phosphorylation in the cells. Most importantly, PHLDA1-silenced cells were less susceptible to apoptosis than control cells, as shown by the lower expression of cleaved caspase-3 and PARP as well as a decreased activity of caspase-3 and -7. Our study negatively correlates expression of PHLDA1 and Aurora A in IMR-32 cells and sheds new light on functions of PHLDA1 in the neuroblastoma tumor cells, suggesting its role as a pro-apoptotic protein. Additionally, our results show possible links of the protein to regulation of features of mitochondria and formation of autophagosomes. IntroductionPHLDA1 was identified as a gene involved in Fas (CD95) expression and apoptosis induced after an anti-TCR antibody binding to mouse T cell hybridoma cells (1). In humans the gene is located on the long arm of the chromosome 12 [12q15 (2)] and encodes a 401 amino acid (aa) protein (45 kDa) named pleckstrin-homology-like domain family A, member 1 (PHLDA1, synonyms include: DT1P1B11, PHRIP, 'prolinehistidine rich protein', TDAG51, based on www.genecards. org).Several research groups have aimed to characterize involvement of PHLDA1 in biology of cancer cells, including its function in apoptosis. Thus, Neef et al measured levels of mRNA of PHLDA1 in three sets of cell lines, derived from paired samples of primary and metastatic melanoma tumors, and reported that PHLDA1 was downregulated in the latter. PHLDA1 was detected by the authors in benign melanocytic nevi, and its level was shown to decrease with progression of malignant melanomas. Also, constitutive expression of the PHLDA1 protein in a melanoma cell line Mel Rif has led to cell growth reduction along with an increase in basal apoptosis and sensitivity to doxorubicin and camptothecin (3). In 2006, Hayashida et al showed that PHLDA1 is a heat shock inducible gene regulated by HSF1 and when overexpressed in HeLa cells has pro-apoptotic functions (4). In yet another report, Nagai et al have reported that downregulation of PHLDA1 is a strong predictor of ...

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Section: Methodsmentioning

confidence: 99%

“…Cells were cultured as described in detail (17). the 14g2a mab was purified from Fbs (fetal bovine serum) free cell culture supernatant as described in detail (17).…”

Section: Methodsmentioning

confidence: 99%

Section: Lentiviral Transduction Imr-32 (25x10mentioning

confidence: 99%

Section: Lentiviral Transduction Imr-32 (25x10mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

See 3 more Smart Citations

Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Durbas

Horwacik

Boratyn

et al. 2016

International Journal of Oncology

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Targeting glycosphingolipids for cancer immunotherapy

Hung

Wang

et al. 2020

FEBS Letters

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Aberrant expression of glycosphingolipids (GSLs) is a unique feature of cancer and stromal cells in tumor microenvironments. Although the impact of GSLs on tumor progression remains largely unclear, anticancer immunotherapies directed against GSLs are attracting growing attention. Here, we focus on GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and Globo H ceramide (GHCer), the most prevalent cancer-associated GSL overexpressed in a variety of epithelial cancers. We first summarize recent advances on our understanding of GD2 and GHCer biology and then discuss the clinical development of the first immunotherapeutic agent targeting a glycolipid, the GD2-specific antibody dinutuximab, its approved indications, and new strategies to improve its efficacy for neuroblastoma. Next, we review ongoing clinical trials on Globo H-targeted immunotherapeutics. We end with highlighting how these studies provide sound scientific rationales for targeting GSLs in cancer and may facilitate a rational design of new GSL-targeted anticancer therapeutics.

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Monocyte Chemoattractant Protein-Induced Protein 1 Overexpression Modulates Transcriptome, Including MicroRNA, in Human Neuroblastoma Cells

et al. 2015

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The recently discovered MCPIP1 (monocyte chemoattractant protein-induced protein 1), a multidomain protein encoded by the MCPIP1 (ZC3H12A) gene, has been described as a new differentiation factor, a ribonuclease, and a deubiquitination-supporting factor. However, its role in cancer is poorly recognized. Our recent analysis of microarrays data showed a lack of expression of the MCPIP1 transcript in primary neuroblastoma, the most common extracranial solid tumor in children. Additionally, enforced expression of the MCPIP1 gene in BE(2)-C cells caused a significant decrease in neuroblastoma proliferation and viability. Aim of the present study was to further investigate the role of MCPIP1 in neuroblastoma, using expression DNA microarrays and microRNA microarrays. Transient transfections of BE(2)-C cells were used for overexpression of either wild type of MCPIP1 (MCPIP1-wt) or its RN-ase defective mutant (MCPIP1-ΔPIN). We have analyzed changes of transcriptome and next, we have used qRT-PCR to verify mRNA levels of selected genes responding to MCPIP1 overexpression. Additionally, protein levels were determined for some of the selected genes. The choline transporter, CTL1, encoded by the SLC44A1 gene, was significantly repressed at the specific mRNA and protein levels and most importantly this translated into a decreased choline transport in MCPIP1-overexpressing cells. Then, we have found microRNA-3613-3p as the mostly altered in the pools of cells overexpressing the wild type MCPIP1. Next, we analyzed the predicted targets of the miR-3613-3p and validated them using qRT-PCR and western blot. These results indicate that the expression of miR-3613-3p might be regulated by MCPIP1 by cleavage of its precursor form.

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Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells

Cited by 34 publications

References 44 publications

Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Downregulation of the PHLDA1 gene in IMR-32 neuroblastoma cells increases levels of Aurora A, TRKB and affects proteins involved in apoptosis and autophagy pathways

Targeting glycosphingolipids for cancer immunotherapy

Monocyte Chemoattractant Protein-Induced Protein 1 Overexpression Modulates Transcriptome, Including MicroRNA, in Human Neuroblastoma Cells

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