The rat adrenal pheochromocytoma cells PC12 (FH0415), mouse hippocampal neuron cells HT22 (FH1027) and human neuroblastoma cells SK-N-SH (FH0164) were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Shanghai, China). PC12 cells were cultured in RPMI-1640 medium (KGM31800, KeyGEN, Nanjing, China) supplemented with 10% fetal bovine serum (FBS, A6901FBS, Invigentech, United States) and 1% penicillin and streptomycin (C0222, Beyotime, Shanghai, China). HT22 and SK-N-SH cells were cultured in DMEM medium (KGM12800, KeyGEN, Nanjing, China) with 10% FBS and 1% penicillin and streptomycin. All cells were incubated in a 37°C with 5% CO₂ atmosphere.

Entrectinib (HY-12678) was obtained from MedChemExpress (MCE, Shanghai, China) with the purity of 99.87%. THBS1 overexpression plasmids were synthesized by Corues Biotchnology (Nanjing, China). The relevant materials used in this study were provided as following: EnoGeneCell Counting Kit-8 (CCK-8, E1CK-000208, EnoGene, Nanjing, China), BeyoClick™ EdU-555 cell proliferation detection kit (C0075S, Beyotime, Shanghai, China), Annexin V-FITC/PI apoptosis detection kit (KGA107, KeyGEN, Nanjing, China), 4% paraformaldehyde (PFA, BL539A, Biosharp, Hefei, China), crystal violet (C0121, Beyotime, Shanghai, China), Trizol (R0016, Beyotime, Shanghai, China), Freezol reagent (R711-01, Vazyme, Nanjing, China), Evo M-MLV RT mix kit (AG11728, Accurate, Changsha, China), AceQ Universal SYBR qPCR master mix (Q511-02, Vazyme, Nanjing, China), BCA protein assay kit (P0010, Beyotime, Shanghai, China), RIPA (P00138, Beyotime, Shanghai, China), ECL detection reagent (180-501, Tanon, Shanghai, China), lipofectamine 3000 transfection reagent (L3000015, Invitrogen, United States).

The antibodies used in Western blotting assay were obtained as following: THBS1 (1:1,000, 18304-1-AP, Proteintech, Wuhan, China), PI3K (1:600, 20584-1-AP, Proteintech, Wuhan, China), AKT (1:5,000, 60203-2-AP, Proteintech, Wuhan, China), p-AKT (1:2,000, 4060T, Cell signaling technology, United States), TGF-β (1:3,000, 21898-1-AP, Proteintech, Wuhan, China), GAPDH (1:60,000, 60004-1-Ig, Proteintech, Wuhan, China), HRP-conjugated affinipure goat anti-rabbit IgG (1:5,000, SA00001-2, Proteintech, Wuhan, China) and HRP-conjugated affinipure goat anti-mouse IgG (1:4,000, SA00001-1, Proteintech, Wuhan, China).

To establish entrectinib activated cells, nerve cells (PC12, HT22 and SK-N-SH) were treated with entrectinib for 48 h at the concentrations of 0, 0.5, 1, 2, 5, 10, and 20 μmol/L, respectively. To explore whether entrectinib influences the cellular proliferation ability and apoptosis, we employed entrectinib (2.3, 4.2 and 4.3 μmol/L, respectively) to the PC12, HT22 and SK-N-SH cells for 48 h. To observe the effect of THBS1 on entrectinib activated cells, nerve cells were divided into five groups: the normal control (NC), the overexpression (OE) group transfected with THBS1 overexpression plasmids, the entrectinib group exposed to entrectinib (2.3, 4.2 and 4.3 μmol/L, respectively), the NC + entrectinib group and the THBS1 overexpression group cultured in medium containing entrectinib (2.3, 4.2 and 4.3 μmol/L, respectively) for 48 h.

PC12, HT22 and SK-N-SH cells were plated in 96-well plates at a density of 7 × 10³ cells per well and incubated for 24 h. Subsequently, PC12, HT22 and SK-N-SH cells were treated with entrectinib and THBS1 overexpression plasmids for 48 h described above. After incubation, 10 μL CCK-8 solution was added into each well and incubated at 37°C for 2 h in the dark. Then, a microplate reader (Molecular Devices, Shanghai, China) was used to measure the absorbance at a wavelength of 450 nm.

PC12, HT22 and SK-N-SH cells were seeded into 24-well plates at a density of 3 × 10⁴ cells per well and treated with entrectinib for 48 h. BeyoClick™ EdU-555 cell proliferation detection kit was used for the subsequent experiments. In brief, 20 μmol/L EdU was added into per well for 4 h. Hoechst 33342 was used to stain the nuclei for 10 min in the dark. Next, images were randomly captured by a fluorescence inverted microscope (Zeiss, Germany). Finally, ImageJ was utilized to count the total cell and proliferating cell numbers.

In six-well plates, PC12, HT22, SK-N-SH cells were cultured at a density of about 7 × 10² cells per well and then treated with entrectinib. The cultures were maintained for 14 days or until the number of single-cell clones exceeded 50. After rinsing with phosphate buffered saline (PBS), cells were fixed with 4% paraformaldehyde solution for 30 min. Next, 0.5% crystal violet solution was stained for 15 min. Finally, cells were washed several times with PBS and captured by a camera.

PC12, HT22 and SK-N-SH cells were seeded into 6-well plates and treated with entrectinib and THBS1 overexpression plasmids for 48 h described above. Then, the treated cells were collected and stained with Annexin V-FITC and PI staining solution for 10 min. Following the recommendations of the manufacturer, the cells were analyzed by flow cytometry (BD Biosciences, United States) within 1 h. Data were analyzed by Flowjo software (v.10.8.1).

PC12, HT22 and SK-N-SH cells were treated with entrectinib, respectively. After 48 h incubation, the total RNA was extracted by Trizol reagent and sequenced in Beijing Biomarker Technology Co., LTD. (Beijing, China). Briefly, according to the manufacturer’s instructions, the sequencing library was generated by Hieff NGS ultima dual-mode mRNA library prep kit (Yeasen, Shanghai, China). The libraries were quantified preliminarily with Qubit 3.0 fluorescence quantitative analyzer and sequenced on an Illumina novaseq platform to generate 150 bp paired-end reads. Clean data were obtained by removing reads containing adapter, ploy-N and low-quality from raw data. The analysis of differentially expressed genes (DEGs) between PC12 and HT22 cells was performed by DESeq2 software (Love et al., 2014), while SK-N-SH cells were analyzed by edgeR software (Robinson et al., 2010). Genes with a statistical threshold of false discovery rate (FDR) < 0.05 and |fold change| > 2 were considered to be significantly differentially expressed. Volcano plots, a visualized plots to illustrate DEGs, were generated by bioinformatics software available at https://www.bioinformatics.com.cn/.

Functional enrichment analyses of GO (Ashburner et al., 2000) and KEGG (Kanehisa et al., 2014) in DEGs from PC12 and HT22 cells were performed by the R package ClusterProfiler (v.4.6.2) (Yu et al., 2012). The R package ggplot2 (v.3.4.1) was utilized to visualize and screen biological functions and pathways associated with these DEGs. Enriched GO terms and KEGG pathways were presented to illustrate the analytical outcomes.

GSEA software (v.4.3.2) (Subramanian et al., 2005) was performed to investigate functional enrichment pathways and landmark gene sets in gene expression datasets. Signature gene sets were extracted from the Molecular Signatures Database (MSigDB, available at https://www.gsea-msigdb.org). Enrichment results were considered significant if the normalized enrichment score (NES) > 1 and the nominal P < 0.05 in the entrectinib treatment.

According to the manufacturer’s recommendations, total cellular RNA was extracted by Freezol reagent and reverse-transcribed into cDNA by the Evo M-MLV RT mix kit. The AceQ universal SYBR qPCR master mix kit was used for quantitative real-time PCR. The β-actin was served as the internal control. The expression of the target gene was calculated by the 2^−ΔΔCT method. Primers used in this study were synthesized by Invitrogen Biotechnology Co., Ltd. (Invitrogen, United States), and their sequences were provided in Supplementary Table S1.

Total protein from cells was extracted through RIPA lysate with a mixture of phosphatase inhibitor and protease inhibitor. The concentration of extracted proteins was quantified through BCA protein assay kit. The protein samples were separated through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. After blocking with 5% skim milk for 2 h, the PVDF membranes were incubated with the primary antibody overnight at 4°C. Subsequently, membranes were incubated with the corresponding secondary antibody and visualized by an enhanced chemiluminescence (ECL) reagent. Protein band intensities were analyzed by ImageJ software.

All experiments were conducted at least in triplicate to ensure reliability. Statistical analysis and graphical representation were carried out by GraphPad Prism (v.9.0). Differences in quantitative variables between two groups were assessed by the t-test, while differences among three or more groups were evaluated by one-way analysis of variance (ANOVA). P < 0.05 was considered as statistical significance.

Initially, the nerve cells (PC12, HT22 and SK-N-SH) were subjected to various concentrations of entrectinib (0, 0.5, 1, 2, 5, 10, and 20 μmol/L) for 48 h, and the impact of entrectinib on nerve cells viability was assessed by the CCK-8 assay. The results indicated that entrectinib significantly inhibited nerve cells viability compared with the control group, displaying a clear dose-dependent reduction trend (Figures 1A, C, E). Subsequently, the IC₅₀ of entrectinib in PC12, HT22 and SK-N-SH cells was calculated as 2.3 μmol/L, 4.2 μmol/L, and 4.3 μmol/L, respectively (Figures 1B, D, F). Thus, the IC₅₀ entrectinib incubation was selected for subsequent experiments. Collectively, these results preliminarily indicated that entrectinib showed an inhibitory effect on the nerve cells viability.

To further investigate entrectinib’s impact on nerve cell proliferation, we performed colony formation and EdU incorporation assays. As shown in Figures 2A–C, the relative colony number of nerve cells (PC12, HT22, SK-N-SH) was significantly decreased to 49.65%, 54.34%, and 50.21% after entrectinib treatment. In addition, EdU incorporation assay demonstrated that the red fluorescence had a notable reduction in the treatment of entrectinib, indicating that entrectinib could inhibit the replicative capacity of nerve cells (Figures 2D–F). These findings confirmed that entrectinib significantly inhibited the proliferation ability of nerve cells.

Moreover, Annexin V-FITC/PI apoptosis detection kit was employed to further explore the impact of entrectinib on nerve cells apoptosis. PC12, HT22 and SK-N-SH cells were treated with entrectinib of 2.3 μmol/L, 4.2 μmol/L and 4.3 μmol/L, respectively, and the apoptosis rate was significantly increased to 12.52%, 14.83% and 15.84% (Figures 3A–D). Taken together, these results indicated that entrectinib could significantly inhibit the viability and induce apoptosis within the nerve cells.

To further explore the impact of entrectinib on nerve cells transcription levels, we employed high-throughput sequencing technology on the entrectinib-treated nerve cells (PC12, HT22 and SK-N-SH) samples. Differential expression analysis revealed that 413, 338, and 481 DEGs were in PC12, HT22 and SK-N-SH cells, respectively (Figures 4A–C; Supplementary Table S2). We then determined the intersection of DEGs present in the nerve cells, where 4 DEGs (FTL1, THBS1, FTL1-PS1, COL3A1) were intersected (Figure 4D). Notably, thrombospondin-1 (THBS1) was significantly downregulated in entrectinib treatment, implying that it might be involved in nerve cell damage (Figure 4E). These findings confirmed that entrectinib could induce extensive transcriptomic changes, and THBS1 was the most significant.

Next, we conducted GO and KEGG pathway enrichment analyses to uncover the biological functions and pathways associated with DEGs. GO results revealed that DEGs were involved in biological processes including peptide cross-linking and epithelial cell proliferation regulation. Meanwhile, it also showed the relationship with cellular components and molecular functions, such as extracellular matrix, transcription repressor complex, growth factor activity and extracellular matrix binding (Figure 5A; Supplementary Table S3). Futhermore, KEGG analysis implicated that DEGs were enriched in pathways such as PI3K-AKT signaling pathway, TGF-β signaling pathway and p53 signaling pathway, et al. (Figure 5B; Supplementary Table S4). Similarly, GSEA was also performed across hallmark gene sets to identify potential signatures of response. The results showed that PI3K-AKT-mTOR signaling pathway, TGF-β signaling pathway and p53 signaling pathway (Figure 5C; Supplementary Table S5) were enriched in entrectinib treatment. The above results suggested that the entrectinib-induced nerve cell damage may be related to the PI3K-AKT-mTOR signaling pathway, p53 signaling pathway and TGF-β signaling pathway.

To validate the transcriptome sequencing analysis results, we further performed qRT-PCR and Western blotting assays. Compared with the control group, the expression of THBS1 was significantly decreased after entrectinib treatment within the nerve cells (Figures 6A, B), which was in parallel with sequencing results. Additionally, the levels of relative proteins involved in PI3K-AKT and TGF-β signaling pathways were examined as well. Obviously, entrectinib could downregulate the expressions of PI3K, AKT, phosphorylated AKT (p-AKT) and TGF-β1 proteins within the nerve cells (PC12, HT22 and SK-N-SH) (Figures 6B–C). Therefore, these results demonstrated that entrectinib could downregulate THBS1 expression while also inhibiting PI3K-AKT and TGF-β signaling pathways.

To preliminary observe and validate whether THBS1 overexpression could rescue nerve cell damage and the abnormalities in PI3K-AKT and TGF-β signaling pathways, nerve cells were transfected with THBS1 overexpression plasmids. As shown in Figures 7A–E, THBS1 overexpression can rescue nerve cell damage induced by entrectinib. Additionally, the expressions of PI3K, AKT, phosphorylated AKT (p-AKT) and TGF-β1 proteins were also upregulated by THBS1 overexpression in the entrectinib-treated cells (Figures 7F–H). These above results suggested that THBS1 plays an important functional role in rescuing nerve cell damage and the abnormalities in the PI3K-AKT and TGF-β signaling pathways induced by entrectinib.