Tamibarotene promotes differentiation of neuroblastoma SH-SY5Y cells into neurons, which is associated with activation of the PI3K/AKT signaling pathway

Abstract

Tamibarotene, a synthetic retinoid used in the treatment of acute promyelocytic leukemia, has been reported to induce differentiation in the SH-SY5Y cell line into neurons. However, the underlying mechanisms remain unclear. This study aimed to determine the optimal concentration of Tamibarotene (Am80) for promoting neuronal differentiation and to elucidate the underlying molecular mechanisms. SH-SY5Y cells were treated with Am80 at various concentrations, and the effects on cell morphology, gene expression, cell proliferation and apoptosis assessed using immunofluorescence, Western blotting, qPCR, and RNA sequencing. Results indicated that that 1µM Am80 effectively promoted neuronal differentiation, upregulating neuronal markers and the KCNT1 gene, while downregulating tumor-related genes MYC and CXCR4. The differentially expressed genes are predominantly enriched in the PI3K-Akt signaling pathway, with upregulation of genes related to neuronal development such as NTRK2, RET, and CNR1, and downregulation of tumor-related genes including MYC and CXCR4. Inhibition of the PI3K/Akt signaling pathway using LY294002 resulted in a decreased efficacy of AM80-induced differentiation in SH-SY5Y cells, along with downregulation of neuronal marker expression. These findings suggest that Am80 can effectively promote the differentiation of SH-SY5Y cells into neurons and reduce the proliferation of neuroblastoma cells, which is related to the PI3K/AKT pathway, providing a good model for the study of nervous system diseases.

Keywords: KCNT1; Cell differentiation; PI3K/Akt signaling pathway; SH-SY5Y; Tamibarotene.

Fig. 1

Cell morphological showed that drug-treated group exhibited slightly smaller cell bodies with extended neurites. The scale bar is 100 μm

Fig. 2

A–B The expression of the neuronal marker NFM increased after Am80 treatment; The control group, n = 6; The 1‰ DMSO group, n = 6; The Am80 1 µM group, n = 7; The scale bar is 50 μm. C The qPCR results revealed that the expression of SYP and KCNT1 genes increased (n = 3); **p < 0.01, ****p < 0.0001

Fig. 3

The RNA sequencing analysis. A Differentially expressed genes were shown in volcano plot. B The bubble plot shows the KEGG pathway enrichment analysis of DEGs in. C DEGs enriched in PI3K-AKT signaling pathway. D Gene Ontology (GO) enrichment analysis of DEGs. E The circle plot of GO enrichment terms related to neuronal differentiation is presented in. F Additionally, a chord plot shows the DEGs enriched in GO terms related to neuronal differentiation. GO:0010976, positive regulation of neuron projection development. GO:0007411, axon guidance. GO:0098978, glutamatergic synapse. GO:0043025, neuronal cell body. GO:0048843, negative regulation of axon extension involved in axon guidance. GO:0001755, neural crest cell migration. GO:0043065, positive regulation of apoptotic process. GO:0042127, regulation of cell population proliferation. (n = 3 per group)

Fig. 4

Am80 activated the PI3K/Akt signaling pathway. A The graph shows the densitometric value of the target protein normalized against the internal reference protein GAPDH n = 4. B Immunofluorescence images showed that PI3K inhibitors significantly inhibited the promotion of SH-SY5Y differentiation into neurons by Am80. C After Am80 treatment, the level of the activated signal p-AKT protein in the PI3K-AKT signaling pathway was significantly increased. And the PI3K inhibitor LY294002 can block Am80-induced expression of neuronal markers. One-way ANOVA followed by Tukey’s post hoc test. *p < 0.05; **p < 0.01; *** p < 0.001

Fig. 5

Am80 inhibited cell proliferation and induced apoptosis. A Cell number-time curve. n = 3. B CCK8-cell proliferation assay. n = 9 from 3 independent experiments. C Cell apoptosis detection. The apoptotic cells were identified by positive green fluorescence, while the living cells showed significantly positive red fluorescence. Scale bar = 100 μm. And the relative expression of Annexin V-FITC in the Am80-treated (n = 11) and control group (n = 5). D Ki-67 was evaluated by immunohistochemistry staining. The proportion of Ki-67 positive cells was significantly reduced in the Am80-treated group was significantly increased. n ≥ 12 per groups. Scale bar = 50 μm. (*p < 0.05, **p < 0.01)