Dyngo-4a Induces Neuroblastoma Cell Differentiation Through The AKT and ERK1/2 Pathway

Abstract

Aim: The aim of the study is to check whether dyngo-4a can inhibit neuroblastoma (NB) proliferation and induce NB cell differentiation Background: Dynamin plays a role in regulating neurotransmission, signaling pathways, nutrient uptake, and pathogen infection, enhancing cell proliferation, tumor invasion, and metastasis. Studies have reported that dyngo-4a, a dynamin inhibitor, can be used to identify potential biomarkers and promising novel therapeutic targets for cancer treatment.

Objective: To our knowledge, no published reports are showing that dynamin inhibitors can reduce NB cell proliferation and induce differentiation. In this study, we report that dyngo-4a can inhibit NB proliferation and induce NB cell differentiation.

Methods: In this study, mouse neuroblastoma (Neuro-2a) cells were cultured in the presence or absence of dyngo-4a or retinoic acid (RA), or in the presence of both dyngo-4a and RA, or in the presence of sequential administration of dyngo-4a and RA to compare the effects on the inhibition of cell proliferation and effects on neuroblastoma cell differentiation induction. The neural cell markers, Nestin and Tuj 1 (Neuron-specific class III beta-tubulin), were used to demonstrate that the differentiated cells have neuronal cell features. The phosphorylation of Protein Kinase B (AKT), extracellular signalregulated kinases1/2 (ERK1/2), and epidermal growth factor receptor (EGFR) were determined to examine the potential mechanisms of induced differentiation.

Results: Dyngo-4a or RA or dyngo-4a with subsequent RA administration induced Neuro-2a cell differentiation. However, RA with subsequent dyngo-4a administration results in almost total death of the Neuro-2a cells. The differentiation rate induced by dyngo-4a was significantly higher than the rate by RA treatment (72.5 ± 1.4% vs. 52.9 ± 3.1% with neuron features, P<0.05; 39.0 ± 0.8% vs. 29.9 ± 1.8% for axons under light microscopy, p<0.05). The differentiation rate of cells treated with dyngo-4a first, followed by RA, was greater than when they were added together (74.8 ± 3.8% vs. 10.6 ± 3.6%; 45.5 ± 1.6% vs. 12.4 ± 0.6%, p<0.01). Co-administration of dyngo-4a and RA at the same time diminished differentiation efficacy significantly. Dyngo-4a induced Neuro-2a cell differentiation and increased Tuj-1 positive staining by the 6^th day post- treatment. Dyngo-4a also inhibited Neuro-2a cell proliferation in a dose-dependent manner. Regarding the mechanism, dyngo-4a treatment showed a significant increase in p-AKT and p-ERK1/2 but not in p-EGFR.

Conclusion: At a level comparable to RA, dynamin inhibition with dyngo-4a lowers proliferation and causes differentiation of Neuro-2a mouse NB cells in vitro. The AKT pathway is activated by dynago- 4a, which results in differentiation. The combination of RA with dynago-4a reduces the efficiency of differentiation. The application of dynago-4a followed by RA, on the other hand, enhances the differentiating effect, implying alternative mechanistic roles in the process.

Keywords: AKT; Dyngo-4a; ERK1/2 pathway; cell proliferation; pathways; retinoic acid.