Abstract

   (1) Background: OSU-2S is a derivative of FTY720 and exhibits
   significant inhibitory effects on various cancer cells. There is
   currently no research on the mechanism of the impact of OSU-2S on NSCLC
   development. We analysed and validated the hub genes and
   pharmacodynamic effects of OSU-2S to treat NSCLC. (2) Methods: The hub
   genes of OSU-2S for the treatment of NSCLC were screened in
   PharmMapper, genecard, and KM Plotter database by survival and
   expression analysis. The effect of OSU-2S on hub gene expression was
   verified by Western blot analysis. The ex vivo and in vivo efficacy of
   OSU-2S on tumour growth was verified using A549 cells and a xenografted
   animal model. (3) Results: A total of 7 marker genes for OSU-2S
   treatment of NSCLC were obtained. AURKA and S1PR1 were screened as hub
   genes. Significant differences in the expression of AURKA and S1PR1
   between normal and lung adenocarcinoma (LUAD) tissues were found in the
   GEPIA2 database; Western blot showed that OSU-2S could affect p-AURKA
   and S1PR1 protein expression. OSU-2S significantly inhibited tumour
   growth in A549 cells and xenografted animal models. (4) Conclusions:
   Our study confirms the inhibitory effect of OSU-2S on NSCLC, screens
   and demonstrates its potential targets AURKA(p-AURKA) and S1PR1, and
   provides a research basis for treating NSCLC with OSU-2S.

   Keywords: OSU-2S, NSCLC, tumor models, bioinformatics analysis, Western
   blot

1. Introduction

   Lung cancer is the most prevalent cancer globally and kills
   approximately 350 people each day, which is 2.5 times the number of
   people who die from rectal cancer. Lung cancer claims the lives of
   around 130,000 individuals annually all around the world [[38]1].
   Small- cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC)
   are included in this cancer. Lung adenocarcinoma (LUAD) and squamous
   lung cancer (LUSC) are the two histologic subtypes of NSCLC, which is
   more common in comparison to SCLC [[39]2]. Lung adenocarcinoma is the
   most prevalent, and its incidence rate accounts for approximately 50%
   of lung cancer cases. The use of targeted agents for treating lung
   adenocarcinoma has gradually become one of the most popular therapeutic
   approaches in the last decade [[40]3,[41]4]. Over 50% of patients with
   stage I or II non-small-cell lung cancer undergo surgery, in comparison
   to just 21% of patients with stage III non-small-cell lung cancer,
   while almost 61% of patients receive chemotherapy and/or radiotherapy
   [[42]5]. Related studies have found that surgery is the more commonly
   used treatment for early-stage NSCLC, sometimes combined with other
   treatments such as chemotherapy, targeted agents, immunotherapy, and/or
   radiation therapy. Advanced NSCLC is generally treated with
   chemotherapy, targeted agents, and/or immunotherapy [[43]6]. As
   early-stage NSCLC is normally asymptomatic, just 30% of cases are
   diagnosed during the first stage and the majority of cases are in
   advanced stages by the time of diagnosis [[44]7]. Therefore, drug
   therapy for advanced NSCLC is essential. The potential targets and
   related mechanisms of new targeted anti-tumour drugs for lung
   adenocarcinoma treatment urgently require basic research.

   FTY720, the generic name Fingolimod, is an FDA-approved sphingosine
   immunosuppressant that targets the sphingosine-1-phosphate (S1P)
   receptor for treating relapsing multiple sclerosis. It is extracted
   from Cordyceps [[45]8,[46]9]. In addition, FTY720 has been reported to
   have anti-tumour effects on a range of cancer types, including breast,
   bladder, liver, and ovarian cancers [[47]10]. According to related
   research, FTY720 activates the ROS/PKCδ-signalling pathway in
   hepatocellular carcinoma cells, which in turn mediates the inactivation
   of caspase-3 [[48]11,[49]12]. To avoid the immunosuppressive effect of
   FTY720, a non-immunosuppressive FTY720 derivative OSU-2S was created by
   structural modification [[50]13,[51]14]. In comparison to FTY720,
   OSU-2S maintained FTY720’s capacity to increase PP2A activity and cause
   PKCδ breakage and caspase-dependent cell death [[52]15]. This provides
   a mechanistic basis for OSU-2S to become an excellent anti-tumour drug.
   The structure of FTY720 and OSU-2S is shown in [53]Figure 1.

Figure 1.

   [54]Figure 1
   [55]Open in a new tab

   Structure of FTY720 and OSU-2S.

   The specific mechanism of the action of the new non-immunosuppressive
   antitumour drug OSU-2S is not very clear. Previous relevant in vitro
   and in vivo studies have found OSU-2S to have obvious cytotoxicity in
   acute lymphocytic leukaemia (ALL), chronic lymphocytic leukaemia (CLL),
   mantle cell lymphoma (MCL), and other diseases, as it induces the death
   of cancer cells [[56]16]. A study of canine spontaneous b-cell lymphoma
   found that OSU-2S induced reactive oxygen species (ROS) production in
   canine lymphoma cells, leading to apoptosis [[57]14]. Research on the
   targets and mechanisms of OSU-2S in anti-tumour treatment requires
   further study. Therefore, this paper analysed and validated the
   potential targets of OSU-2S action with NSCLC by utilising network
   pharmacology and bioinformatics methods.

2. Results

2.1. Acquisition of Marker Genes

   One hundred OSU-2S effector targets were predicted in the Swiss
   TargetPrediction database, and 299 OSU-2S effector targets were
   predicted in the PharmMapper database. 334 effector targets remained
   after the two were combined and duplicates were removed. 1050 and 160
   NSCLC targets were obtained from Genecard database and TTD database,
   respectively. The two were merged to remove duplicate values to obtain
   1159 targets, which were analyzed by PPI and uploaded to
   Cytoscape_v3.7.2 software to screen the top 700 genes by the 4 modules
   in the cytoHubba plug-in. The intersection was taken in order to obtain
   564 disease targets ([58]Figure 2A). The [59]GSE10072 dataset was
   corrected ([60]Figure 2B) for differentially expressed gene analysis,
   and 1624 DEGs were obtained, including 750 up-regulated genes and 874
   down-regulated genes. [61]Figure 2C,D displays the heatmap and volcano
   diagram of differentially expressed genes (DEGs). Seven marker genes
   were obtained by taking the intersection of DEGs, OSU-2S targets, and
   disease genes ([62]Figure 3A,B).

Figure 2.

   [63]Figure 2
   [64]Open in a new tab

   Acquisition of genes associated with lung adenocarcinoma. (A) cytoHubba
   plugin was used to screen for NSCLC disease genes. (B) Expression
   corrected PCA analysis of [65]GSE10072 data matrix, dark green
   represents normal tissue, light yellow represents tumor tissue. (C)
   Heat map of differentially expressed genes, with red areas showing
   positive correlation and blue areas showing negative correlation. (D)
   Differentially expressed gene volcano plot, dark green represents
   up-regulated genes and light yellow represents down-regulated genes.

Figure 3.

   [66]Figure 3
   [67]Open in a new tab

   Screening and analysis of marker genes. (A,B) Venn diagrams of the
   three intersecting genes, DEGs, OSU-2S targets of action and disease
   targets, with gene expression box line plots. *** represents p < 0.001.

2.2. Survival and Expression Analysis of Marker Genes

   The results of survival analysis of the marker genes found that 6 genes
   (PARP1, CDK4, FGFR2, AURKA, NR3C1, and S1PR1) had significant
   prognostic significance in NSCLC. However, MMP9 was not statistically
   significant in the prognostic analysis of NSCLC ([68]Figure 4A). The
   results of the mRNA expression analysis of the marker genes in LUAD and
   LUSC found AURKA to be significantly up-regulated and S1PR1 to be
   significantly down-regulated in LUAD and LUSC ([69]Figure 4B). On the
   basis of the results of the two analyses, AURKA and S1PR1 were chosen
   as hub genes.

Figure 4.

   [70]Figure 4
   [71]Open in a new tab

   Survival and expression analysis of marker gene. (A) KM survival
   analysis of marker gene. (B) Expression analysis of marker in lung
   adenocarcinoma and lung squamous carcinoma, light red represents tumour
   and grey represents normal. * represents p < 0.05.

2.3. Functional Enrichment of Hub Genes

   Enrichment analysis of the single-gene GSEA-KEGG pathway ([72]Figure 5)
   found that AURKA and S1PR1 are all involved in DNA replication signals
   and can affect the cytochrome P450 enzyme system in drug metabolism,
   which suggests that hub genes affect tumour progression during the DNA
   replication phase of tumour cell growth while also having the potential
   for involvement in tumour drug resistance. In addition, AURKA are
   involved in the functional pathway of the one-carbon unit donor folate,
   S1PR1 directly affects protein output, and S1PR1 plays roles in amino
   acid synthesis. These results suggest that hub genes could be involved
   in multiple key signalling mechanisms in tumour growth and may be
   crucial in tumour drug resistance.

Figure 5.

   [73]Figure 5
   [74]Open in a new tab

   Single-gene signaling pathway analysis. Single gene GSEA-KEGG pathway
   enrichment analysis of AURKA and S1PR1.

2.4. OSU-2S Effectively Inhibited the Cell Viability, Migration, and Colony
Formation of A549 Cells

   To verify the effect of OSU-2S on lung adenocarcinoma cells during
   growth, A549 cells were selected for administration by different
   concentrations of OSU-2S for 24 h. The CCK-8 results showed OSU-2S to
   have the ability to inhibit A549 cell proliferation ([75]Figure 6A,B).
   Based on the CCK-8 assay results, we confirmed by clone formation and
   wound-healing assay experiments that OSU-2S-treated A549 cells
   exhibited lower migration and clone formation ability ([76]Figure
   6C,F).

Figure 6.

   [77]Figure 6
   [78]Open in a new tab

   A549 cells were treated with OSU-2S for CCK-8, clone formation assays,
   and wound-healing assays. (A,B) A549 cells were treated with different
   concentrations OSU-2S for 24 h before the cell viability was determined
   via CCK8. (C,D) Crystalline violet staining to analyze colony formation
   in A549 cells treated by three different concentrations (1.5 × 10^−6, 3
   × 10^−6 and 6 × 10^−6 mol/L) of OSU-2S. All values are expressed as the
   mean ± SD (n = 3). (E) A549 cells were treated with three different
   concentrations (1.5 × 10^−6, 3 × 10^−6 and 6 × 10^−6 mol/L) OSU-2S for
   24 h, and the intercellular spacing was observed at different time
   points (0 h, 12 h, and 24 h). (F) The statistical analyses of the
   migration ability. All values are expressed as the mean ± SD (n = 3).
   Scale bar, 50 μm. * represents p < 0.05, *** represents p < 0.001 and
   **** represents p < 0.0001.

2.5. Validation of AURKA, S1PR1 Protein Expression

   To confirm OSU-2S’s impact on lung adenocarcinoma cells during growth,
   A549 cells were selected for administration by different concentrations
   of OSU-2S for 24 h. The results showed that OSU-2S has the ability to
   inhibit A549 cell proliferation in a dose-dependent manner.
   Furthermore, the effect of OSU-2S on the differentially expressed
   proteins AURKA, p-AURKA, and S1PR1 was further verified by Western
   blotting. The results showed that different OSU-2S concentrations (1.5
   × 10^−6, 3 × 10^−6, and 6 × 10^−6 mol/L) could significantly inhibit
   the p-AURKA and S1PR1 expressions with the increase in the
   concentration after treating A549 cells for 24 h, as can be seen in
   [79]Figure 7A–D. These results confirmed that OSU-2S had a significant
   regulatory effect on p-AURKA and S1PR1 expressions in A549 lung
   adenocarcinoma cells, and the effect on AURKA expression was not
   obvious. However, the specific signalling pathways that are affected by
   the regulation of p-AURKA and S1PR1 expressions require further
   in-depth investigation, which our next study will focus on.

Figure 7.

   [80]Figure 7
   [81]Open in a new tab

   Effect of OSU-2S on the expression of AURKA, S1PR1 detected by western
   blotting assay A549 cells were exposed to three different
   concentrations (1.5 × 10^−6, 3 × 10^−6, and 6 × 10^−6) of OSU-2S for 24
   h. (A) Effect of different concentrations of OSU-2S on the expression
   of AURKA and S1PR1 protein. (B–D) The statistical analyses of the
   protein expression. All values are expressed as the mean  ±  SD (n =
   3). * represents p < 0.05.

2.6. OSU-2S Showed Anti-Tumour Activity in a Mouse Lung Cancer Model

   An A549 xenograft lung cancer mouse model was used for evaluating the
   anti-tumour effect of OSU-2S. A549 cells were injected into the axilla
   of mice as a means of producing primary tumours at the injection site.
   Different doses of OSU-2S were then administered when the tumour volume
   reached 50 mm^3. The tumours were isolated subcutaneously from the mice
   after 35 days of treatment and then weighed (n = 6). In comparison to
   the solvent (β-hydroxypropyl-cyclodextrin), the tumour volume and total
   weight were considerably reduced by OSU-2S at 5 and 10 mg/kg
   ([82]Figure 8A–C). Additionally, the mice’s weight did not
   significantly decrease ([83]Figure 8D), which indicated that OSU-2S at
   the dosage of 10 mg/kg is well tolerated in BALB/c nude mice.

Figure 8.

   [84]Figure 8
   [85]Open in a new tab

   Anti-tumour effect of OSU-2S on A549 xenograft lung cancer mice. (A)
   Tumours were isolated subcutaneously from mice after 35 days of
   treatment (n = 6). (B) Tumour volume was measured weekly during the
   experiment (n = 6). (C) Tumour weights were measured after 35 days of
   treatment (n = 6). (D) Weights of mice were measured weekly (n = 6)
   during treatment with β-hydroxypropyl-cyclodextrin, FTY720, and OSU-2S
   (2.5 mg/kg, 5 mg/kg, 10 mg/kg). * represents p < 0.05, ** represents p
   < 0.01 and **** represents p < 0.0001.

2.7. Immunohistochemical Detection of Vascular Endothelial Growth Factor and
Tumor Proliferation Markers

   Vascular endothelial growth factor (VEGF) expression is closely
   associated with angiogenesis in tumour tissues and lung cancer growth.
   Increased VEGF uexpression can increase tumour tissue angiogenesis,
   promoting lung cancer growth. Therefore, the determination of VEGF
   expression using immunohistochemical methods and the determination of
   tumorigenicity based on proliferation markers (Ki-67) are essential for
   the detection of the anti-tumorigenic capacity of drugs in vivo. OSU-2S
   significantly downregulated the expression levels of Ki-67 and VEGF in
   tumour tissues and inhibited tumour growth ([86]Figure 9A–C). The
   results suggest that OSU-2S has a definite inhibiting effect on the
   development of lung cancer in vivo.

Figure 9.

   [87]Figure 9
   [88]Open in a new tab

   The effect of OSU-2S on the expression of proliferation and apoptosis
   markers in tumour tissues. (A) Representative IHC images of VEGF and
   Ki67 of the tumors. (B,C) Quantification analysis of immunoreactive
   cells for VEGF and Ki67. Data are presented as mean  ±  SD (n  =  3).
   Scale bar, 50 μm. ** represents p < 0.01, *** represents p < 0.001 and
   **** represents p < 0.0001. ns not significant.

3. Discussion

   The most prevalent kind of NSCLC is LUAD [[89]17], and it develops
   abnormally from type II alveolar cells of the airway epithelium that
   secrete mucus and other substances [[90]2]. It is the main culprit
   responsible for mortality linked to lung cancer in individuals. In
   contrast to other lung cancer subtypes, LUAD exhibits a gradual
   progression and lacks apparent clinical signs during its initial
   stages. Approximately 30–75% of patients are already in an advanced
   phase when they receive diagnosis, by which time radical treatment is
   impossible [[91]18]. Therefore, early detection and treatment of LUAD
   are crucial [[92]19]. The exploration of potent early tumour indicators
   during routine examinations has emerged as a prominent aspect in LUAD
   investigations, and the discovery and application of drugs that target
   markers is also key to LUAD treatment. OSU-2S is a derivative of FTY720
   that also has inhibitory effects on a variety of tumour cells, and
   following structural modification, it was found that OSU-2S was not
   phosphorylated, thereby avoiding the immunosuppressive effect of FTY720
   [[93]13,[94]14]. In the current era of increasing cancer disease, the
   new anticancer drug OSU-2S is highly expected in tumour therapy, but
   current basic research on the anticancer effect of OSU-2S is lacking
   and this is an urgent problem that requires solving. Therefore, this
   study utilized network pharmacology and bioinformatics methods to
   obtain potential targets for the treatment of NSCLC by OSU-2S, verified
   the efficacy of OSU-2S on NSCLC and its regulatory effects on potential
   targets through in vivo and in vitro experiments, and provided a
   reliable theoretical foundation for the early detection and treatment
   of LUAD.

   In this study, two genes (AURKA and S1PR1) were screened as hub genes
   of OSU-2S treatment NSCLC by survival analysis and expression analysis,
   and the study has confirmed the tumour-growth-suppressive effect of
   OSU-2S in in vivo and in vitro experiments.

   AURKA, AURKB, and AURKC are the three members of the serine-threonine
   kinase family. AURKA and AURKB play essential roles in cell division,
   proliferation, and migration through the regulation of the cell mitosis
   process [[95]20]. A related study found that AURKA acts as an oncogene
   to facilitate the promotion of the growth of many tumour types, such as
   solid tumours and haematologic malignancies [[96]21,[97]22,[98]23]. A
   related study also found that knockdown AURKA substantially escalated
   the effectiveness of radiation on HCT116 and HT-29 cell proliferation.
   The combined impact of AURKA inhibition and radiation demonstrated a
   potent capacity to suppress cellular migration and metastasis while
   also instigating apoptosis synergistically [[99]24]. Relevant
   bioinformatics analysis suggests AURKA may be a key gene in lung
   cancer, which could lead to poor prognosis [[100]25]. Furthermore,
   CXCL5, which is a key downstream of AURKA, was demonstrated to be
   suppressed after AURKA silencing in NSCLC. The AURKA-CXCL5 axis is
   critical to regulate the radiosensitivity of NSCLC in the cell
   autophagy process [[101]26]. Bioinformatics analysis in this study
   highlighted the enhanced expression of AURKA in LUAD tissues, which was
   consistent with the above findings. The OSU-2S treatment of A549 cells
   resulted in minimal changes in total AURKA protein, while the
   expression of p-AURKA appeared to be significantly reduced in
   comparison to the controls. The above results suggest that p-AURKA is
   potentially a key marker for LUAD growth and metastasis, and it may
   also be a potential target of OSU-2S for LUAD treatment.

   S1PR1 (Sphingosine-1-phosphate receptor 1) is a G protein-coupled
   receptor that is responsible for the mediation of the effects of the
   biologically active sphingosine 1-phosphate (S1P). S1PR1 is a member of
   a subfamily of receptors known as sphingosine-1-phosphate receptors.
   These receptors are crucial in the maintenance of the structural
   integrity of endothelial cells, facilitating cell migration, modulating
   immune responses, and promoting angiogenesis [[102]27]. Related studies
   have found S1PR1 to exhibit pronounced expression in malignant bladder
   cells, which correlates with an unfavourable prognosis among affected
   individuals [[103]28]. S1P was also found to be able to enhance cancer
   cell viability and promote cancer cell growth and metastasis by binding
   to S1PR1 [[104]29]. Furthermore, according to related research, S1PR1
   regulates lymphocyte proliferation and differentiation inside the
   cancer microenvironment [[105]30]. It was also reported that genetic
   deletion of S1P receptor 1 (S1PR1) in macrophage-infiltrated mouse
   mammary tumours prevents lung metastasis and tumour lymphangiogenesis
   [[106]31]. In lung-cancer-related studies, it was found that the
   proliferation and invasion of NSCLC cells in vitro and tumor growth in
   vivo could be promoted by up-regulating the expression of S1PR1. In
   addition, inhibition of S1PR1 can be achieved by targeting inhibited
   NSCLC cell proliferation, migration, and invasion, while inducing more
   NSCLC cell apoptosis [[107]32,[108]33]. We identified S1PR1 as an
   anticancer target with potential research value in LUAD by network
   pharmacology. It was also further confirmed that OSU-2S can
   down-regulate the expression of S1PR1 in A549 cells in protein
   immunoblotting experiments. However, the specific signalling pathway
   through which OSU-2S affects S1PR1 requires further investigation.

   Western blotting analysis confirmed that OSU-2S could down-regulate
   p-AURKA and S1PR1 expressions in A549 cells. GSEA analysis showed AURKA
   and S1PR1 to be involved in various important biological cell growth
   processes, including one-carbon unit donor folate function, DNA
   replication signalling, and amino acid synthesis, which may be the key
   to the anticancer effect of OSU-2S. Studies on anticancer drugs have
   demonstrated that methotrexate interferes with folate metabolism as a
   means of blocking the utilisation of one carbon unit in tumour cells,
   which inhibits cell division. Cisplatin is a common drug used to treat
   non-small-cell lung cancer, and its mechanism of action is to stop the
   DNA replication of tumour cells and block cell division to produce an
   anti-tumour effect [[109]34,[110]35,[111]36]

4. Materials and Methods

4.1. Data Sources

   The chemical structure of OSU-2S was obtained from the PharmMapper
   ([112]http://www.lilab-ecust.cn/pharmmapper/, accessed on 10 October
   2022) database for identifying its target. The targets of OSU-2S were
   searched in the Swiss TargetPrediction
   ([113]http://www.swisstargetprediction.ch/, accessed on 10 October
   2022) database using Canonical SMILES of OSU-2S as the keyword. (The
   number of OSU-2S targets obtained was small, so targets were not
   screened based on relevant conditions.) The R GEO query package
   [[114]37] was used for obtaining the original dataset [115]GSE10072
   from the high-throughput Gene Expression Omnibus, (GEO)
   ([116]https://www.ncbi.nlm.nih.gov/geo/, accessed on 23 July 2023)
   database in the [117]GPL96 ([HG-U133A] Affymetrix Human Genome U133A
   Array) platform as the experimental set for analysis (including 58 lung
   adenocarcinoma tissues and 49 normal lung tissues). To better match the
   lung adenocarcinoma genes in the [118]GSE10072 dataset, disease
   databases at Genecard ([119]https://www.genecards.org/, accessed on 2
   August 2023) and TTD ([120]http://db.idrblab.net/ttd/, accessed on 20
   August 2023) were searched for disease targets with “lung
   adenocarcinoma” used as the keyword (gene screening conditions were
   greater than or equal to the 4-fold median of correlation score).

4.2. Screening of Disease Genes and Differentially Expressed Genes

   Disease genes obtained from the Genecard and TTD databases were merged,
   and duplicate values were removed. Before being filtered using the
   cytoHubba plugin in Cytoscape_v3.7.2, taking the intersection of the
   genes ranked in the top 700 (the genes ranked higher in the cytoHubba
   plug-in make more significance) in the four modules of Betweenness,
   Closeness, Degree, and Stress. The intersection of the top 700 genes
   was defined as the disease genes. The dataset that was used in this
   study was corrected by NormalizeBetweenArrays to ensure the removal of
   the batch effect, and the differentially expressed genes (DEGs) between
   healthy lung tissues and adenocarcinoma tissues was examined employing
   the R software (Version 4.3.1) limma package [[121]38] with |log2FC| >
   0.5 and p < 0.05 as the criteria to determine DEGs. The criteria of
   log2FC > 0.5 and p < 0.05 were used to identify differentially
   expressed genes (DEGs). The intersecting genes of OSU-2S targets,
   disease genes, and DEGs were obtained as marker genes for OSU-2S
   inhibition of NSCLC by Using the jvenn diagram tool
   ([122]https://jvenn.toulouse.inra.fr/app/example.html, accessed on 20
   August 2023) and Microsoft Excel.

4.3. Survival Analysis and Expression Analysis of Marker Genes

   The marker gene survival analysis was carried out employing the KM
   Plotter database ([123]http://kmplot.com/analysis/, accessed on 19
   September 2023). Employing the GEPIA2 database, expression analysis of
   the marker genes in lung adenocarcinoma and lung squamous carcinoma was
   carried out ([124]http://gepia2.cancer-pku.cn/, accessed on 19
   September 2023). The results of the two analyses were integrated, and
   whether the p-value was significantly different was used as a screening
   condition for hub genes (p < 0.05 was considered to be significantly
   different).

4.4. GSEA-KEGG Enrichment and Protein Expression Analysis of Hub Genes

   Correlations between hub genes and other genes in the [125]GSE10072
   dataset were analysed by the R software GSEA package. The KEGG
   signalling pathway set was then called a predefined set for the
   detection of the enrichment of hub genes in the signalling pathway set.

4.5. Cell Experiments

4.5.1. Cell Culture

   Lung adenocarcinoma A549 cell lines (American Type Culture
   Collection(ATCC), Manassas, VA, USA) were cultured in F12K (Procell,
   Wuhan, China) medium supplemented with 100 mg/mL streptomycin (TRANS,
   Beijing, China) and 100 U/mL penicillin (TRANS, Beijing, China), 10%
   fetal bovine serum (FBS) (Sigma-Aldrich, Burlington, MA, USA), under 37
   °C, 5% CO[2] and high humidity in a constant temperature incubator.

4.5.2. Cell Counting Kit (CCK)-8 Assay

   The Cell Counting Kit-8 (CCK-8) (Solarbio, Beijing, China) was used to
   evaluate the cell viability according to the manufacturer’s
   instructions (Bioss, Beijing, China). Cell culture conditions were as
   described in [126]Section 4.5.1, and A549 cells (3 × 10^3 cells/well)
   were incubated in 96-well plates for 24 h. The CCK-8 solution (10 μL)
   was added to each well, and the plates were incubated for 1 h at 37 °C;
   then the absorbance at 450 nm wavelength (OD 450) was measured in a
   microplate reader (Biorad, Hercules, CA, USA).

4.5.3. Cell Migration

   Cell culture conditions were as described in [127]Section 4.5.1, and
   well-grown A549 cells were inoculated into 6-well plates at 1 × 10^−6
   cells per well and placed in an incubator at 37 °C, 5% CO[2] for 24 h
   in order to form a monolayer of cells the following day. The tip of a
   1000 μL micropipette was used for scratching the monolayer of cells to
   ensure that a uniform scratch was formed in the monolayer. The cells
   were rinsed as a means of removing cellular debris and incubated in 1%
   FBS medium with or without different concentrations of OSU-2S (1.5 ×
   10^−6, 3 × 10^−6 and 6 × 10^−6 mol/L) for 24 h. Images were taken of
   the wells every 6 h using light microscope observation. All the data
   were analysed using Image-J software((Version 1.8.0).

4.5.4. Colony Formation Assay

   An in vitro clone formation assay was used for measuring colony
   formation. Cell culture conditions were as described in [128]Section
   4.5.1, and A density of 200 cells per well was inoculated into a 6-well
   plate and placed in an incubator at 37 °C with 5% CO[2] for overnight
   incubation in order to ensure adhesion. The cells were incubated in the
   medium with or without different concentrations of OSU-2S (1.5 × 10^−6,
   3 × 10^−6, and 6 × 10^−6 mol/L) the following day for 14 days.
   Following the conclusion of the incubation period, the cells were
   washed twice with PBS (Procell, Wuhan, China). Four percent
   paraformaldehyde solution was used for fixation for 30 min, and the
   cells were stained with crystal violet dye for 10 min at room
   temperature. The cells were then washed using deionised water and were
   allowed to dry overnight before the number of different clones in each
   condition were counted. The experiment was repeated three times.

4.5.5. Western Blotting

   Cell culture conditions were as described in [129]Section 4.5.1, and
   6-well plates were used to seed A549 cells (5 × 10^5 cells/well). The
   cells were incubated for 12 h before being exposed to OSU-2S for 24 h.
   Following cell harvesting, entire cells were lysed in RIPA solution in
   order to extract all of the proteins. Before being transferred to a
   polyvinylidene fluoride membrane (PVDF; Millipore, Boston, MA, USA)
   sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After that,
   the membrane was washed with 1×TBST (Solarbio, Beijing, China) solution
   for 3 times, 5 min each time. The membranes were sealed for an hour at
   room temperature using 5% skim milk. The membrane was subjected to an
   overnight 4 °C incubation with the primary antibody (anti-AURKA,
   anti-S1PR1 at 1:1000; Affinity, San Francisco, CA, USA), and the
   membrane was washed with 1×TBST solution for 3 times, 5 min each time.
   An hour-long room temperature incubation was carried out with the
   horseradish peroxidase-conjugated secondary antibody (Affinity, San
   Francisco, CA, USA), and the membrane was washed with 1×TBST solution
   for 3 times, 5 min each time. Bound proteins were visualized using a
   chemiluminescence imaging system (Biorad, Hercules, CA, USA).

4.6. Animal Experimentation

4.6.1. Experimental Mouse

   Male BALB/c nude mice (4-6 weeks old, body weight 20 ± 2 g) were
   purchased from Charles River and placed in a controlled experimental
   environment conforming to spf grade standards in the Animal
   Experimentation Center of Xinjiang Medical University, at a temperature
   of 23 ± 2 °C, with standard food and sterile drinking water, and a
   light/dark cycle of 12 h. The present study was approved by the Ethics
   Committee for Experimental Animals of Xinjiang Medical University
   (Ethics The study was approved by the Laboratory Animal Ethics
   Committee of Xinjiang Medical University (Ethics Approval No.
   IACUC-JT-20230423-20).

4.6.2. Establishment of a Xenograft Mouse Model and Grouping

   After the mice were acclimatized, well-conditioned A549 cells (1 ×
   10^7) were injected subcutaneously into the axilla. When the tumour
   diameter reached 50 mm^3, the mice were randomly assigned to five
   groups (n = 6) and given intraperitoneal injections of FTY720 (5
   mg/kg), OSU-2S (2.5 mg/kg, 5 mg/kg, and 10 mg/kg groups) and
   β-hydroxypropyl-cyclodextrin as a control once every other day for 35
   days. The growth of the tumour and the body weight of the mice was
   recorded on a weekly basis. The formula used to determine the tumour
   volume was as follows: volume = length × width^2 × 1/2.

4.6.3. Immunohistochemistry of Tumour Tissue

   The mice’s tumour tissues were fixed for 24 h with a 4%
   para-formaldehyde solution (Solarbio, Beijing, China) after being
   cleaned with saline. They were then dehydrated and embedded in paraffin
   wax. The slices were baked at 60 °C for 12 h, placed in a slice box,
   and stored in the refrigerator at 4 °C for future use. The sections
   were dewaxed and then incubated with goat serum and an endogenous
   peroxidase blocker. After that, the slices were incubated with
   anti-Ki67 (Affinity, San Francisco, CA, USA) and anti-VEGF (Affinity,
   San Francisco, CA, USA) antibodies for an entire night at 4 °C. After
   incubating the sections with DAB and a goat anti-rabbit secondary
   antibody (Affinity, San Francisco, CA, USA), the slices were examined
   and photographed under a microscope.

4.7. Statistical Analysis

   R 4.3.1 was utilised to conduct the processing and analysis of the data
   in the bioinformatics portion of the study, and the Wilcoxon rank sum
   test was employed to compare the two groups. The GraphPad Prism 8.0
   (comparisons between groups were performed using one-way analysis of
   variance (ANOVA)) and ImageJ software were used for the statistical
   analysis of CCK-8, Western blotting, and immunohistochemistry results.
   All the experiments in this study were independently repeated a minimum
   of three times: p < 0.05 represents significant difference.

5. Conclusions

   In conclusion, our study provides strong experimental evidence that
   OSU-2S has a significant anti-tumour effect in vitro and in vivo. Two
   hub genes (AURKA and S1PR1) were screened as early markers of LUAD
   pathogenesis and potential targets for OSU-2S to exert anti-LUAD by
   network pharmacology and bioinformatics methods. It was found that
   OSU-2S may down-regulate the expression of S1PR1 and AURKA, which may
   prevent the growth of cancer cells. However, the exact molecular
   mechanism of action requires more thorough in vitro and in vivo
   experiments to be properly determined. The next study will focus on the
   molecular pathways that are involved in AURKA and S1PR1 as a means of
   providing more feasible options for LUAD treatment.

Acknowledgments