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In recent years, immunotherapy has been identified as an effective treatment method for high-risk neuroblastoma. A previous study demonstrated that an anti-GD2 ganglioside (GD2) mouse 14G2a monoclonal antibody (mAb) combined with a small molecule, i.e., an aurora A kinase inhibitor (MK-5108), significantly increased cytotoxicity against human neuroblastoma cells, as compared to monotherapy. This study aimed to demonstrate the mechanism of neuroblastoma cell death in vitro following the addition of an anti-GD2 human-mouse chimeric ch14.18/CHO mAb (presently used in clinics) and two aurora A inhibitors (MK-5108 and MK-8745). The effects of the aforementioned agents on neuroblastoma cells were determined by measuring the level of ATP, the level of apoptotic and necroptotic markers, and the activity of caspase 3/7. The results revealed that the ch14.18/CHO mAb decreased cellular ATP levels in the IMR-32 and CHP-134 neuroblastoma cell lines, similarly to the 14G2a mAb. Regarding ch14.18/CHO mAb treated IMR-32 cells, the observed cytotoxic effect was concomitant with induced caspase 3 cleavage, which indicated the induction of apoptosis in IMR-32 cells, but not in CHP-134 cells. Furthermore, the MK-5108 inhibitor induced apoptosis, as indicated by the increased cleavage of caspase 3 and increased activity of caspase 3/7. However, the presence of necroptosis was ruled out in MK-5108-treated IMR-32 and CHP-134 cells. In summary, the effects of the combination of ch14.18/CHO mAb and aurora A kinase inhibitors (MK-5108 and MK-8745) were shown to enhance apoptosis in IMR-32 cells compared to when used individually.

The process of autophagy and its role in survival of human neuroblastoma cell cultures was studied upon addition of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (14G2a mAb) and an aurora A kinase specific inhibitor, MK-5108. It was recently shown that combination of these agents significantly potentiates cytotoxicity against IMR-32 and CHP-134 neuroblastoma cells in vitro, as compared to the inhibitor used alone. In this study we gained mechanistic insights on autophagy in the observed cytotoxic effects exerted by both agents using cytotoxicity assays, RT-qPCR, immunoblotting, and autophagy detection methods. Enhancement of the autophagy process in the 14G2a mAb- and MK-5108-treated IMR-32 cells was documented by assessing autophagic flux. Application of a lysosomotropic agent—chloroquine (CQ) affected the 14G2a mAb- and MK-5108-stimulated autophagic flux. It is our conclusion that the 14G2a mAb (40 μg/ml) and MK-5108 inhibitor (0.1 μM) induce autophagy in IMR-32 cells. Moreover, the combinatorial treatment of IMR-32 cells with the 14G2a mAb and CQ significantly potentiates cytotoxic effect, as compared to CQ used alone. Most importantly, we showed that interfering with autophagy at its early and late step augments the 14G2a mAb-induced apoptosis, therefore we can conclude that inhibition of autophagy is the primary mechanism of the CQ-mediated sensitization to the 14G2a mAb-induced apoptosis. Although, there was no virtual stimulation of autophagy in the 14G2a mAb-treated CHP-134 neuroblastoma cells, we were able to show that PHLDA1 protein positively regulates autophagy and this process exists in a mutually exclusive manner with apoptosis in PHLDA1-silenced CHP-134 cells. Electronic supplementary material The online version of this article (10.1007/s10495-018-1472-9) contains supplementary material, which is available to authorized users.

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 protein 1 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 factor 1 (APAF1), Dicer, DNA fragmentation factor subunit β, von Hippel‑Lindau protein and neurofibromin 1] 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.

The importance of monocyte chemotactic protein-1-induced protein 1 (MCPIP1) in the negative regulation of inflammatory reactions has already been extensively studied. However, its role in cancer is not yet established. We studied MCPIP1 gene expression in primary human neuroblastomas and several neuroblastoma cell lines. Our results showed a lack of MCPIP1 expression in primary neuroblastoma tumors. Moreover, it was found that the low expression of the protein measured in human neuroblastoma cell lines might be important for neuroblastoma survival, since enforced MCPIP1 gene expression in human neuroblastoma BE(2)-C cells caused a significant decrease in neuroblastoma cell viability and proliferation.

The main physiological function of MCPIP1 (regnase-1) is negative regulation of inflammation. Moreover, roles of regnase-1 in apoptosis and differentiation have also been described, but its involvement in cancer is yet to be fully recognized. Earlier, we showed a lack of expression of MCPIP1 in both primary tumors and several neuroblastoma cell lines. Additionally, we reported that levels of MCPIP1 and the key neuroblastoma oncoprotein-MYCN were inversely correlated in BE(2)-C clones overexpressing the MCPIP1 gene. Here, we show that exogenous expression of the MCPIP1 protein decreases MYCN mRNA and protein levels without changing the MYCN mRNA half-life. Furthermore, it was shown that MCPIP1-wt exogenous expression affects levels and phosphorylation of MYCN partners such as Aurora A (Thr288), CDC2 (Tyr15 and Thr161), GSK3β (Ser9), and key cellular components of Akt/mTOR signaling, which regulate MYCN stability and activation. In accordance with the obtained results, we found increased phosphorylation of MYCN protein at Thr58 that causes destabilization of the oncoprotein. Moreover, it is shown that exogenous expression of MCPIP1 does not cause apoptosis. Our data extend knowledge on roles of MCPIP1 in our model and link the protein to regulation of expression and stability of MYCN through decrease of signaling via Akt/mTOR pathway. J. Cell. Biochem. 118: 1741-1755, 2017. © 2016 Wiley Periodicals, Inc.

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.

Sulforaphane (SFN) is an isothiocyanate present in cruciferous vegetables, which has been shown to exert an anti-cancer effect when tested in vitro and in vivo. The anti-cancer effects of SFN encompass induction of cytoprotective autophagy; therefore, the present study aimed to determine whether the chemopreventive activity of SFN may be potentiated by inhibition of autophagy. The present study provided detailed insight into the susceptibility of human neuroblastoma cells to treatment with synthetic SFN, in combination with an inhibitor of autophagy, 3-methyladenine (3-MA). The present study confirmed the suppression of the viability of the human neuroblastoma cell line BE(2)-C by SFN and reported the inhibition of DNA synthesis, as determined by a decrease in tritiated thymidine incorporation. Furthermore, the results verified the effectiveness of SFN in inducing apoptosis in the BE(2)-C cell line as demonstrated by caspase activation, increased protein expression levels of B-cell lymphoma 2-associated X protein and loss of mitochondrial membrane potential. Combined treatment of the cells with SFN with 3-MA proved to be effective in decreasing cell viability, through a mechanism that may proceed via the early induction of autophagy by SFN, followed by induction of apoptosis, as well as inhibition of autophagy by 3-MA.

Mechanisms leading to inhibitory effects of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) and PI3K/Akt/mTOR pathway inhibitors on human neuroblastoma cell survival were studied in vitro. We have recently shown on IMR-32, CHP‑134, and LA-N-1 neuroblastoma cells that targeting GD2 with the mAb decreases cell viability of the cell lines. In this study we used cytotoxicity assays, proteomic arrays and immunoblotting to evaluate the response of the three cell lines to the anti‑GD2 14G2a mAb and specific PI3K/Akt/mTOR pathway inhibitors. We show here that the mAb modulates intracellular signal transduction through changes in several kinases and their substrates phosphorylation. More detailed analysis of the PI3K/Akt/mTOR pathway showed significant decrease in activity of Akt, mTOR, p70 S6 and 4E-BP1 proteins and transient increase in PTEN (a suppressor of the pathway), leading to inhibition of the signaling network responsible for stimulation of translation and proliferation. Additionally, combining the GD2-specific 14G2a mAb with an Akt inhibitor (perifosine), dual mTOR/PI3K inhibitors (BEZ-235 and SAR245409), and a pan-PI3K inhibitor (LY294002) was shown to enhance cytotoxic effects against IMR-32, CHP‑134 and LA-N-1 cells. Our study extends knowledge on mechanisms of action of the 14G2a mAb on the neuroblastoma cells. Also, it stresses the need for further delineation of molecular signal orchestration aimed at more reasonable selection of drugs to target key cellular pathways in quest for better cure for neuroblastoma patients.