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. Graphical abstract [38]graphic file with name 12868_2025_962_Figa_HTML.jpg Supplementary Information The online version contains supplementary material available at 10.1186/s12868-025-00962-8. Keywords: Tamibarotene, SH-SY5Y, Cell differentiation, KCNT1, PI3K/Akt signaling pathway Introduction Tamibarotene (Am80) is a synthetic CYP26-resistant retinoid known for its high specificity for retinoic acid receptors α and β [[39]1]. It is utilized as a treatment for acute promyelocytic leukemia (APL). Recent clinical investigations have looked into the potential of AM80-based therapy for relapsed/refractory acute myeloid leukemia (AML) with overexpression of the retinoic acid receptor alpha (RARα) gene [[40]1, [41]2]. A study suggested that tamibarotene may mitigate sepsis-induced lung injury by deregulating the NF-κB signaling pathway [[42]3]. Additionally, Am80 has also been demonstrated to stimulate the cardiomyocyte cell cycle and enhance engraftment in the heart by activating endogenous Wnt pathways [[43]4]. The SH-SY5Y cell line is one of the most commonly used cell models in neuroscience research. It is a thrice-cloned subline of the SK-N-SH cell line, which was derived from a metastatic bone marrow biopsy taken from a 4-year-old girl diagnosed with neuroblastoma [[44]5, [45]6]. Under normal culture conditions, the SH-SY5Y cell line proliferated as adherent neuroblast-like cells. However, this cell line has the capability to differentiate into neurons when exposed to various inducing agents, such as retinoic acid (RA), histone deacetylase (HDAC) inhibitors, lactate, B-27, and others [[46]7–[47]10]. RA activates nuclear receptors like retinoid receptors and retinoid X receptors to modulate gene transcription [[48]11]. Previous studies have shown that compared with the traditional retinoids, tamibarotene is chemically more stable [[49]12] and can more effectively induce neuronal differentiation in SH-SY5Y cells [[50]13]. However, the main mechanism by which Am80 induces differentiation of SH-SY5Y cells into mature neurons remains unclear. In this study, we aimed to investigate the mechanism of Am80 as an inducing agent for SH-SY5Y cells and its effect on transcriptome changes. Our findings demonstrate that Am80 promotes SH-SY5Y cell differentiation mainly through PI3K/AKT signaling pathway, while down-regulating corresponding pathways in cancer. Transcriptomic analysis revealed that Am80 inhibits the expression of proto-oncogenes and upregulates the expression of genes related to neuronal differentiation, including MYC, CXCR4, NTRK2, RET and CNR1. The results indicate that 1 µM Am80 can effectively induce the differentiation of SH-SY5Y cells into neurons by activating the PI3K/Akt signaling pathway, providing a reliable model for neuroscience research. Additionally, it can reduce the expression of tumor-associated genes and inhibit tumorigenicity, suggesting that Am80 may be a potential therapeutic approach for solid tumors. Materials and methods Cell culture and treatment In our cellular experiments, we adopted randomization and blinding procedures to ensure the reliability and validity of our results. The cell populations were randomly assigned into separate experimental and control groups. The treatment administrations were carried out in a randomized manner, rather than in a fixed sequence, to minimize potential confounding variables associated with temporal or environmental biases. Data collection was performed by individuals who were unaware of the group assignments, thus eliminating any observational bias that might arise from prior knowledge of the experimental conditions. To confirm the reproducibility and reliability of our outcomes, each experimental model was independently replicated at least three times. The SH-SY5Y cell line (RRID: CVCL_0019) was generously provided by Professor Yun Wang of the Neuroscience Research Institute, Peking University. The SH-SY5Y cells were cultured in DMEM/F12supplemented with 10% fetal bovine serum and 1% Penicillin–Streptomycin solution at 37 ℃ with 5% CO[2] and a humidity of 95% in incubators. Cells were passaged at 70%–80% confluency using 2.5% Trypsin in a 37 ℃ incubator for 1 min and then inactivated by DMEM/F12 + 10%FBS + 1%PS. Tamibarotene was purchased from Sigma Corporation and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, USA). The initial experiment was divided into 5 groups: control group, 1 ‰ DMSO group, 0.1 µM Am80 group, 1.0 µM Am80 group, and 10µM Am80 group. The medium was replaced completely every 1–2 days. Under the Olympus CKX41 inverted microscope with a fixed field of view, morphological observations and photo records were conducted on the day 0, day 1, day 3, day 5, and day 7, respectively. Unless otherwise indicated, cells were cultured in 6-well plates at a density of 200,000 cells per well. Cell counts are performed using the FIJI software. Western blot After 7 days of treatment in different groups, cells were washed three times with ice-cold PBS. Subsequently, they were incubated in ice-cold RIPA lysis buffer (C1055, APPLYGEN) for 10 min on ice. The lysis products were collected using 1.5 ml EP tubes and centrifuged at 12,000 rpm for 15 min at 4 ℃. The supernatant contains total cellular protein. Protein concentrations were determined with Bicinchoninic Acid assays (PC0020, Solarbio). All proteins were mixed with the loading buffer (5×), then heated for 5 min at 95 °C. Proteins were separated with 8% SDS-PAGE gel and transferred onto PVDF membranes using an iBlot-2 system (25 V, 6 min). Membranes were blocked with a solution of 5% nonfat powdered milk, and then incubated overnight at 4 °C with rabbit anti-neurofilament medium ([51]AB254348, CST, 1:1000), rabbit anti Phospho-Akt (4060T, CST, 1:1000), rabbit anti Akt (9272 S, CST, 1:1000) and mouse anti-GAPDH primary antibodies (CSB-MA000071M1m, CUSABIO, 1:1000). After this step was completed, membranes underwent another round of incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for one hour. Finally, the protein bands were visualized by enhanced chemiluminescence detection reagents. Before all steps, wash membranes with 1x TBS-T 3 times for 10 min each time. Protein expression levels (relative to GAPDH expression) were quantified using FIJI software. Immunofluorescent staining SH-SY5Y cells were cultured in a 20-mm Glass Bottom Cell Culture Dish (NEST) precoated with 0.1 mg/mL Poly-D-lysine (P7280, Sigma–Aldrich) with a cell density of 100,000. After 7 days, cells were fixed with 4% PFA for 10 min before being permeabilized with 0.3% Triton X-100 for another 10 min. Subsequently, the cells were blocked with 5% bovine serum albumin (ST2249, Beyotime) for 30 min and incubated overnight at 4 °C with primary antibodies, rabbit anti β-III Tubulin (AB18207, Abcam, 1:400) and mouse anti-neurofilament heavy polypeptide (2836 S, CST, 1:400). Following this, the cells were incubated with the appropriate secondary antibodies: goat anti-mouse antibody conjugated with Alexa Fluor-568 (A-11004, Invitrogen) or goat anti-rabbit antibody conjugated with Alexa Fluor-488 (A-11008, Invitrogen) for one hour at room temperature. Nuclei were counter-stained with Hoechst 33 342 (H3570, Invitrogen, 1:2000) for 10 min. Before each step, the cells were washed three times with PBS for 10 min each time. Finally, immunofluorescence imaging was performed using an FV10i confocal laser scanning microscope (Olympus Corporation, Japan). A total of 19 images were captured and have been included in the supplementary materials. Finally, coverslips were examined using an FV10i confocal microscope (Olympus, Japan). 19 images were taken and have been added to the supplementary. FIJI software was used to quantify the mean fluorescence intensity of each group. q-PCR Total RNA was extracted from the cells after 7 days of treatment in different groups using the RNAeasy™ Animal RNA Isolation Kit with spin column (R0024, Beyotime). The RNA was converted to cDNA using the EasyScript^® One-Step gDNA Removal and cDNA Synthesis SuperMix (AE311, TransGen Biotech) according to the manufacturer’s protocol. Quantitative PCR was performed with BeyoFast™ SYBR Green qPCR Mix (D7260, Beyotime). All primers listed in Table [52]1 were purchased from Beijing Tianyi Huiyuan Biotechnology Co, LTD (China). The cycle threshold (Ct) was recorded, and the relative expression of target genes was calculated by the 2^−ΔΔCt method. GAPDH was used as an internal control to normalize the data. Primer sequences were provided in the table below (Table [53]1). Table 1. q-PCR primers used in this study Gene Primer Type Primer sequence5’→3’ SYP Forward Reverse TGGGGACTACTCCTCGTCAG GTGGCCAGAAAGTCCAGCAT KCNT1 Forward Reverse GAGTTTGACGACGGCCAATG CGGATCCTCAGGCTCGATCT GAPDH Forward Reverse GAAAGCCTGCCGGTGACTAA AGGAAAAGCATCACCCGGAG [54]Open in a new tab RNA sequencing analysis TRIzol reagent (15596018, ThermoFisher) was used to extract total mRNA from cells cultured for 7 days in control group and 1 µM Am80 treatment group, and RNA sequencing was performed in Chigene Transformation Medical Research Center. Differential expression analysis of genes between the two conditions was conducted using the DEGseq R software package (version 1.20.0). Differentially expressed genes (DEGs) were defined based on a q-value < 0.05 and the absolute value of log2 Fold Change (|log2FC|) is not less than 2. For annotating potential functions and conducting enrichment analysis, we utilized the Database for Annotation, Visualization, and Integrated Discovery (DAVID, [55]https://david.ncifcrf.gov/) for the gene ontology (GO) and the Kyoto encyclopedia Genes and Genomes (KEGG) analysis. The volcano map and bar graphs were generated using the R Programming Language with ggplot2. The GOBar and GOChord graphs were created using the GOplot package (version 1.0.2) in R Programming Language. CCK-8 assay SH-SY5Y cells (6.0 × 10^3) were seeded in 96-well plates and treated with 1 ‰DMSO, Am80 0.1 µM, Am80 1 µM, and Am80 10 µM for 7 days, respectively. Afterwards, cells were exposed to 10% CCK-8 (C0038, Beyotime) for another 1 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher). Cell proliferation and Annexin V assay SH-SY5Y cells were cultured in a 20-mm Glass Bottom Cell Culture Dish (NEST) precoated with 0.1 mg/mL Poly-D-lysine (P7280, Sigma–Aldrich) with a cell density of 100,000. After 7 days of drug treatment, cell proliferation and apoptosis assay were conducted using a Ki-67 Cell Proliferation Assay Kit (C2301S, Beyotime) and Mitochondrial Membrane Potential and Apoptosis Detection Kit with Mito-Tracker Red CMXRos and Annexin V-FITC (C1071S, Beyotime), respectively. The samples were incubated and then imaged on the FV10i confocal microscope (Olympus, Japan). Data analysis Data were analyzed using GraphPad Prism software (version 8.0) and expressed as mean ± standard deviation (mean ± SD). The Shapiro-Wilk test was employed to evaluate the normality of variance among the groups. The method for determining the homogeneity of variance was the Bartlett’s test or the Brown-Forsythe test. Data that conformed to a normal distribution were subjected to independent sample t-tests for pairwise group comparisons, whereas one-way ANOVA was applied for assessing differences across multiple groups. If the data satisfy the assumption of normality but not the assumption of homoscedasticity, then the Brown-Forsythe and Welch ANOVA tests or the unpaired t-test with Welch’s correction are employed. p-value < 0.05 is onsidered significant. Statistical significance was denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The results of the tests for normality and variance homogeneity are found in Supplementary materials. Results Am80 promoted the differentiation of SH-SY5Y into neurons in vitro A phase contrast microscope was utilized to observe the morphological characteristics and proliferation of SH-SY5Y cells on days 0, 1, 3, and 7 after treatment with Am80 or DMSO (Fig. [56]1). As shown in Fig. [57]1, compared to the control and the 1 ‰ DMSO treated group, the Am80 treatment group exhibited smaller cell bodies with conical or triangular shapes, along with significantly elongated neurites. After 7 days of Am80 treatment, the cell count was lower than that of the controls. Additionally, the differentiation capacity of SH-SY5Y cells treated with both 1 µM and 10 µM Am80 was found to becomparable. Fig. 1. [58]Fig. 1 [59]Open in a new tab Cell morphological showed that drug-treated group exhibited slightly smaller cell bodies with extended neurites. The scale bar is 100 μm Then, we assessed the expression of neuronal markers in SH-SY5Y cells through cell immunofluorescence and qPCR after 7 days of cell culture in the 1 µM Am80 group, the control group, and the 1‰ DMSO group. Cell immunofluorescence revealed significant increases in neuron-specific marker β-III Tubulin and the mature neuron marker neurofilament heavy polypeptide (NFH, p < 0.0001) in the 1µM Am80 treated group compared to the control group (Fig. [60]2A, B). Furthermore, we evaluated the relative expression levels of SYP and KCNT1. SYP encodes an integral membrane protein of small synaptic vesicles in the brain, serving as a neuronal marker. KCNT1 encodes a sodium-activated potassium channel subunit which plays a crucial role in maintaining neuronal excitability. The qPCR results indicated upregulation of the expression of SYP gene (p = 0.004 < 0.05) and KCNT1 gene (p = 0.0015 < 0.05) (Fig. [61]2C). These findings collectively confirm that Am80 promotes the differentiation of SH-SY5Y cells into mature neurons. Fig. 2. [62]Fig. 2 [63]Open in a new tab 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 RNA sequencing analysis To further investigate the potential mechanism of Am80-induced cell differentiation, transcriptome analysis was conducted between the 1 µM Am80 treated group and the 1‰DMSO group. A total of 387 differentially expressed genes (DEGs) were identified, consisting of 260 up-regulated genes and 127 down-regulated genes (shown in Fig. [64]3A). Fig. 3. [65]Fig. 3 [66]Open in a new tab 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) To gain a deeper understanding of the biological functions and pathways associated with these DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. KEGG pathway enrichment analysis revealed that the DEGs showed a significant association with key pathways, including pathways in cancer, PI3K-AKT signaling pathway, Neuroactive ligand-receptor interaction and et al. (Fig. [67]3B). GO enrichment analyses revealed significant associations of the DEGs primarily with “protein binding”, “plasma membrane”, “extracellular space” and “signal transduction”. It is noteworthy that the main enriched GO terms of DEGs are biological pathways, followed by cellular components, whereas the least number of GO terms are related to molecular function (Fig. [68]3C). The GO functional enrichment analysis revealed a total of 8 GO terms related to neuronal differentiation, positive regulation of neuron projection development, axon guidance, glutamatergic synapse, neuronal cell body, negative regulation of axon extension involved in axon guidance, neural crest cell migration, positive regulation of apoptotic process, regulation of cell population proliferation (Fig. [69]3D, E). Tamibarotene activated PI3K/AKT signaling pathway To examine tamibarotene’s effect on the further PI3K/AKT signaling pathway, western blotting and cellular immunofluorescence were employed to determine the expression of neurofilament medium chain (NFM) protein, a marker of mature neurons. In order to verify the role of this pathway in SH-SY5Y differentiation into neurons, based on the previous experimental results, we added 5 µM of the PI3K-specific inhibitor LY294002 in the 1 µM Am80 treatment group. The results showed that after 7 days of experimental treatment, the expression of neurofilament medium chain (NFM) protein, a mature neuronal marker, significantly increased in the SH-SY5Y cells treated with different concentrations of Am80 (p = 0.0009 < 0.001, p = 0.0222 < 0.05, and p = 0.0383 < 0.05, Fig. [70]4A, C). Fig. 4. [71]Fig. 4 [72]Open in a new tab 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 To verify the role of the PI3K/AKT pathway in Am80 -induced neuronal differentiation, this study examined the expression levels of p-AKT and AKT, and added the PI3K inhibitor LY294002 to the 1 µM Am80-treated group. The results showed that compared with the control group, the expression of p-AKT was significantly elevated after a 7-day treatment with 1 µM Am80 (p = 0.0007 < 0.001, Fig. [73]4A, C). In the group with the addition of the PI3K inhibitor LY294002, the expression of the neuronal-specific marker NFM was significantly decreased (p = 0.0239 < 0.05, Fig. [74]4A, C), and the cell protrusions were markedly reduced and shortened (Fig. [75]4B). The results indicate that the PI3K/AKT signaling pathway is a crucial pathway in the process of Am80-induced differentiation of SH-SY5Y cells into neurons. Am80 inhibits cell proliferation and induces apoptosis To evaluate the inhibitory effects of Am80 at various concentrations on the proliferation of SH-SY5Y cells, we conducted time-lapse imaging to monitor changes in cell number over time and performed the CCK-8 proliferation assay. The results indicated that as the duration of drug treatment increased, cell growth in the treated group decelerated. After 5–7 days of treatment, the cell count in the treated group was significantly lower than that in the control and DMSO solvent groups (Fig. [76]5A). And, the CCK-8 proliferation assay was conducted 7 days post-drug treatment. The results showed that on the 7th day of drug treatment, the cell proliferation rate in the Am80-treated groups was significantly lower than that in the DMSO-treated group (1‰ DMSO vs. Am80 0.1 µM, p = 0.0318; 1‰ DMSO vs. Am80 1 µM, p = 0.0057 < 0.05; 1‰ DMSO vs. Am80 10 µM, p = 0.0023 < 0.05). Fig. 5. [77]Fig. 5 [78]Open in a new tab 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) We further evaluated cell proliferation and apoptosis in the control group and the 1.0 µM Am80 treatment group using Ki-67 and Annexin V-FITC immunofluorescence assays. Compared to the control group, the 1 µM Am80 treated group exhibited a significant increase in green fluorescence expression, indicating apoptotic cells (p = 0.0025 < 0.01). Annexin V assay results suggested that Am80 drug treatment increased cell death (Fig. [79]5A–B). After 7 days, SH-SY5Y cells cultured in 1 µM Am80 showed reduced Ki-67 expression, a result that suggested reduced cell proliferation (p = 0.0154 < 0.05, Fig. [80]5C, D). Discussion The SH-SY5Y neuroblastoma cell line is widely utilized as a model for investigating the molecular and cellular processes involved in neurodegenerative disease and drug toxicology studies in vitro [[81]14, [82]15]. It is more accessible and proliferates rapidly compared to primary neurons, and it also offers lower costs and requires less time than stem cell-induced neurons. Previously, RA has been the most commonly used agent for differentiating SH-SY5Y cells into neurons [[83]16]. Tamibarotene (Am80), the medication of interest in this study, is a synthetic anti-CYP26 retinoid used as a novel approach in acute myeloid leukemia [[84]2, [85]17]. Previous study has shown that Am80 can more effectively induce the differentiation of SH-SY5Y cells into neuron-like cells [[86]13]. However, prior research has only validated the differentiation of SH-SY5Y cells using a single neuronal marker, GAP43, without confirming the changes in transcriptional levels or the underlying mechanisms following drug treatment. In our study, we thoroughly evaluated the process of differentiation from SH-SY5Y to neurons induced by Am80 multiple times and established the critical role of the PI3K/AKT signaling pathway in cell differentiation. After 7 days of treatment with Am80, SH-SY5Y cells exhibited elongated protrusions that were interlinked in a network, and differentiated into neuron-like cells expressing neuron-specific cytoskeleton proteins NFH and β-III Tubulin. Neurofilament proteins (Nfs) are composed of five subunits: neurofilament light chains (NFL), neurofilament medium chains (NFM), neurofilament heavy chains (NFH), alpha-internexin (INA), and peripherin (PRPH). These components are the main neuron-specific elements responsible for maintaining structural integrity [[87]18]. The microtubule protein β-III Tubulin, which is encoded by the TUBB3 gene, is the most highly expressed protein in the early stages of exuberant synaptogenesis and plays a crucial role in normal cellular processes such as vesicular transport and cell motility [[88]19]. Therefore, Am80 is able to promote the differentiation of SH-SY5Y cells into neurons, as confirmed by the increased expression of NFH, and β-III Tubulin. In addition, the relative expression of the SYP gene, which encodes synaptophysin regulating neurotransmitter release and synaptic plasticity, was found to be increased in neuron-like cells following induction of Am80 [[89]20]. The KCNT1 gene encodes the Slack channel, which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarization [[90]21], and its relative expression is also upregulated. qPCR results demonstrated that the relative expression of SYP and KCNT1 increased, suggesting that Am80-induced differentiation of neurons can lead to functional changes. To investigate the mechanism of SH-SY5Y cell differentiation induced by Am80 into neurons. After being cultured for 7 days, we conducted transcriptomic analysis to compare the 1‰ DMSO group with the 1 µM Am80 treatment group. The GO enrichment analysis of differentially expressed genes revealed enrichment in GO terms associated with neuronal differentiation, such as glutamatergic synapse, axon guidance, positive regulation of neuron projection development and so on. The NTRK2 gene was found to be up-regulated and enriched in the majority of GO terms. This gene encodes neuronal receptor tyrosine kinase-2 (TrkB), a critical role of neuronal survival, proliferation, differentiation and apoptosis [[91]22]. NTRK2/TrkB is expressed in neuroblastomas with unfavorable evolution, usually accompanied with MYCN amplification [[92]23, [93]24]. The expression of RET gene, which encodes Rearranged during transfection (RET) protein, was significantly up-regulated in Am80 treated group. RET is the tyrosine kinase receptor identified as the receptor of GDNF, which triggers the neurite outgrowth and the survival of the central nervous system neurons [[94]25]. CNR1 coding cannabinoid receptors, a study showed that the receptor is required for neurodevelopment of Striosome-Dendron Bouquets [[95]26]. The up-regulation of these genes proved that Am80 can induce SH-SY5Y cells to differentiate into neuronal cells, and also demonstrated the importance of these genes in nervous system development. Furthermore, KEGG enrichment analysis showed that the DEGs were mainly enriched in ‘Pathway in cancer’ and ‘PI3K/Akt signaling pathway’. Specifically, the differentially expressed genes associated with the PI3K/Akt signaling pathway exhibited higher -Log[10]|P-values|. To verify the role of the PI3K/AKT signaling pathway in the differentiation of SH-SY5Y cells into neurons induced by Am80, we added the PI3K-targeted inhibitor LY294002 to the 1 µM Am80 treatment group. Western blotting results showed that compared with the control group, the levels of neurofilament medium chain (NFM) and phosphorylated protein kinase B (p-Akt) were significantly increased in the Am80-treated group. After the addition of a PI3K-specific inhibitor, the expression of these proteins was markedly reduced, and the cells exhibited shorter and fewerneurites. Previous studies have found that the PI3K/AKT signaling pathway is an essential signaling mechanism that plays a crucial role in regulating cell cycle progression, proliferation, differentiation, migration, and metabolism [[96]27, [97]28]. These findings are consistent with a previous study which suggested that Am80 can regulate apoptosis-related proteins by PI3K/AKT signaling pathway to protect the ischemic hippocampus [[98]29]. Additionally, Tzeng et al. reported that the PI3K/AKT signaling pathway plays an important role in maintaining cell survival during neural differentiation [[99]30]. Our findings suggest that the PI3K/AKT signaling pathway plays a crucial role in the process of SH-SY5Y cells differentiating into neurons. MYCN oncogene amplification is commonly seen in aggressive pediatric neuroblastoma, and the development of specific Myc protein inhibitors is of great significance for the treatment of neuroblastoma [[100]31]. The proto-oncogene MYC was found to be downregulated in SH-SY5Y-induced neurons. The result suggest that Am80 may be effective in treating neuroblastoma. Another downregulated gene CXCR4 has been shown to control organ and tissue regeneration as well as promote tumor growth. It is also associated with poor prognosis in various cancers [[101]32, [102]33]. These findings indicated that tumor-related genes and pathways were down-regulated while neurodevelopment-related genes and pathways were up-regulated after Am80 treatment. Cell morphology analysis revealed a significant reduction in cell count in the Am80 treatment group when compared to the control group. The CCK8 experiment also confirmed that after drug treatment, the cell proliferation rate decreased. Moreover, the expression of Annexin V-FITC increased and the expression of Ki-67 decreased. The results indicated that Am80 could significantly inhibit the proliferation of SH-SY5Y cells and induce cell apoptosis. Previous reports have indicated that pediatric acute myeloid leukemia (pAML) samples exhibit specific sensitivity to Am80 in vitro, characterized by decreased proliferation and the induction of apoptosis [[103]34, [104]35]. The above results suggest that Am80 may be one of the potential therapeutic approaches for neuroblastoma. It provides a possibility for exploring the treatment of neuroblastoma, but whether it can be truly applicable to clinical treatment requires more oncology and pharmacological experiments for verification. Based on the experimental results, we have demonstrated that Am80 can reliably induce differentiation of SH-SY5Y cells into neuronal models. This model may be effectively utilized in future neuroscience and drug toxicology research. Our study also suggests that the PI3K/AKT signaling pathway is primarily involved in promoting neurodifferentiation and inhibiting the proliferation of neuroblastoma cells. Electronic supplementary material Below is the link to the electronic supplementary material. [105]Supplementary Material 1^ (98MB, zip) [106]Supplementary Material 2^ (101MB, zip) [107]Supplementary Material 3^ (55.7MB, zip) [108]Supplementary Material 4^ (27.1MB, zip) Acknowledgements