GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Durbas, Małgorzata; Horwacik, Irena; Boratyn, Elżbieta; Kamycka, Elżbieta; Rokita, Hanna

doi:10.3892/ijo.2015.3070

Cited by 33 publications

(26 citation statements)

References 61 publications

(82 reference statements)

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“…Specifically, its aberrant activation is common in NBL and also correlates with poor prognosis [37, 46, 47]. Thus, the inhibition of PI3K/Akt/mTOR pathway might prove clinically effective in NBL treatment [47].…”

Section: Discussionmentioning

confidence: 99%

RETRACTED ARTICLE: Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment

Giacoppo

Iori

Rollin

et al. 2017

BMC Complement Altern Med

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BackgroundSeveral lines of evidence suggest the consume of natural products for cancer prevention or treatment. In particular, isothiocyanates (ITCs) exerting anti-cancer properties, have received great interest as potential chemotherapeutic agents.This study was designed to assess the anti-proliferative activities of a new preparation of Moringa oleifera-derived 4-(α-L-rhamnopyranosyloxy)benzyl ITC (moringin) complexed with alpha-cyclodextrin (moringin + α-CD; MAC) on SH-SY5Y human neuroblastoma cells. This new formulation arises in the attempt to overcome the poor solubility and stability of moringin alone in aqueous media.MethodsSH-SY5Y cells were cultured and exposed to increasing concentrations of MAC (1.0, 2.5 and 5.0 μg). Cell proliferation was examined by MTT and cell count assays. The cytotoxic activity of the MAC complex was assessed by lactate dehydrogenase (LDH) assay and trypan blue exclusion test. In addition, western blotting analyses for the main apoptosis-related proteins were performed.ResultsTreatment of SH-SY5Y cells with the MAC complex reduced cell growth in concentration dependent manner. Specifically, MAC exhibited a potent action in inhibiting the PI3K/Akt/mTOR pathway, whose aberrant activation was found in many types of cancer. MAC was also found to induce the nuclear factor-κB (NF-κB) p65 activation by phosphorylation and its translocation into the nucleus. Moreover, treatment with MAC was able to down-regulate MAPK pathway (results focused on JNK and p38 expression). Finally, MAC was found to trigger apoptotic death pathway (based on expression levels of cleaved-caspase 3, Bax/Bcl-2 balance, p53 and p21).ConclusionThese findings suggest that use of MAC complex may open novel perspectives to improve the poor prognosis of patients with neuroblastoma.

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Section: Discussionmentioning

confidence: 99%

RETRACTED ARTICLE: Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment

Giacoppo

Iori

Rollin

et al. 2017

BMC Complement Altern Med

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“…Previously, we reported that the increased level of PHLDA1 in IMR-32 cells treated with 14G2a mAb correlated with the decreased level of Aurora A, p-Aurora A (Thr288) and p-akt (thr308) (17,24). also, a specific aurora a inhibitor, MK-5108, affected the PHLDA1 level in a time-dependent manner in the cell line (17).…”

Section: Discussionmentioning

confidence: 99%

“…Our earlier studies showed that treatment of IMR-32 cells with 14G2a led to increase in PHLDA1 levels in IMR-32 cells, accompanied by a decrease in: total levels of MYCN (25), Aurora A and B, activating phosphorylations of Aurora A (Thr288), Aurora B (Thr232), Aurora C (Thr198) (17), Akt (Thr308), and mTOR (Ser2448) in whole cell extracts (24). Thus, we compared the levels of the aforementioned proteins between the S2 and S4 clones and control cells after 48 h of the 14G2a-treatment (Figs.…”

Section: Phlda1 Downregulation Increases Phosphorylation Ofmentioning

confidence: 99%

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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“…Thus, we showed that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 antibody 14G2a (mAb). Furthermore, we have already reported an increase in p53, but a drop of MYCN in nuclear protein fractions, a decrease in Aurora A kinase, and downregulation of activity of the Akt/mTOR signaling network, which correlated with decreased cellular ATP levels in IMR-32 neuroblastoma cells after the mAb treatment (10,11). Also, we were able to show that 13-cis retinoic acid, Aurora A inhibitor MK-5108, a dual PI3K and mTOR inhibitor BEZ-235, when combined with 14G2a, can further decrease cellular ATP levels in some neuroblastoma cell lines (10,11).…”

Section: Modulation Of Interactions Of Neuroblastoma Cell Lines With mentioning

confidence: 69%

“…Wu et al (33) reported that migration of NB8 neuroblastoma cells mediated by α5β1 integrin involved pathways engaging SRC and FAK, but movement mediated by α4β1 integrin involved only SRC. We have already shown that dephosphorylation of β-catenin (phosphorylation of the protein is involved in anoikis resistance) (40), Tyr397 of FAK (the phosphorylation is important for cell survival) (41), an increase in activating phosphorylation of Tyr420 of FYN (expression of active FYN is linked to good outcome in neuroblastoma independently of MYCN amplification) (42), downregulation of AKT/mTOR network, a decrease in nuclear levels of MYCN (a known regulator of FAK expression) (43) are all linked among other changes of proteins to treatment of IMR-32 with 14G2a (10,11). From the available literature, it is known that targeting kinases linked to integrin signaling can be exploited in neuroblastoma therapy.…”

Section: Discussionmentioning

confidence: 99%

Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a

Horwacik

Rokita

2017

International Journal of Oncology

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Children diagnosed with high risk neuroblastoma have poor prognosis which stimulates efforts to broaden therapies of the neoplasm. GD2-ganglioside (GD2) marks neuroblastoma cells and is a target for monoclonal antibodies. We have recently shown that some neuroblastoma cell lines are sensitive to direct cytotoxicity of the anti-GD2 mouse monoclonal antibody 14G2a (mAb). For IMR-32 and LA-N-1 cell lines, treatment with the 14G2a mAb induced evident changes in appearance such as cell rounding, aggregation, loose contact with culture plastic, or detachment. Such findings prompted us to investigate whether modulation of attachment of neuroblastoma cells to extracellular matrix (ECM) proteins can affect their sensitivity to the 14G2a mAb treatment. First, using ultra-low attachment plates, we show that survival of the IMR-32, LA-N-1, LA-N-5, CHP-134 and Kelly cells depends on attachment. Next, we compared cellular ATP levels of the cell lines treated with the 14G2a mAb using uncoated, fibronectin-, collagen IV-coated surfaces to show that the ECM proteins slightly modulate sensitivity of the cell lines to the mAb. Then, we characterized presence of selected integrin subunits or their complexes on the cell surface. Finally, we applied small molecule inhibitors of selected integrin complexes: obtustatin (inhibiting α1β1 heterodimer), BIO 1211 (inhibiting active α4β1 heterodimer), cilengitide and SB273005 (inhibitors of αVβ3, αVβ5 heterodimers) to verify their effects on attachment of cell lines, cellular ATP levels, and in some experiments activities of apoptosis-executing caspase-3 and -7, for the compounds used alone or in combination with the 14G2a mAb. We characterized levels of total FAK (focal adhesion kinase), p-FAK (Tyr397) in IMR-32 cells treated with BIO 1211, and in LA-N-5, Kelly and SK-N-SH cells treated with SB273005. Our results extend knowledge on factors influencing cytotoxicity of 14G2a.

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GD2 ganglioside specific antibody treatment downregulates PI3K/Akt/mTOR signaling network in human neuroblastoma cell lines

Cited by 33 publications

References 61 publications

RETRACTED ARTICLE: Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment

RETRACTED ARTICLE: Moringa isothiocyanate complexed with α-cyclodextrin: a new perspective in neuroblastoma treatment

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

Modulation of interactions of neuroblastoma cell lines with extracellular matrix proteins affects their sensitivity to treatment with the anti-GD2 ganglioside antibody 14G2a

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