Network Pharmacology Analysis
Prediction of Drug Targets for α-solanine
Molecular file of α-solanine were downloaded from Pubchem (https://pubchem.ncbi.nlm.nih.gov/). Then the targets of the constituents were collected from PharmMapper (http://www.lilab-ecust.cn/pharmmapper/submitfile.html). The obtained targets were imported into UniProt (https://www.uniprot.org/) to search their official gene.
Collection And Analysis Of Gene Targets For Nsclc
The genes of NSCLC patients were obtained from the TCGA database (https://portal.gdc.cancer.gov). Differentially expressed gene analysis was performed in R software. Genes with a P value < 0.05 and |log2(fold change) | > 2 were considered to be of significantly differential expression between NSCLC tissue samples and normal samples. The R package “DEseq2” was used for differential expression analysis. The heatmap and volcano plot were drawn with “pheatmap” R package and “ggplot2” R package.
Screening of candidate targets and construction of network
The intersection targets of α-solanine and NSCLC were obtained by Venn tool(http://bioinformatics.psb.ugent.be/webtools/Venn/), namely the candidate genes of α-solanine for NSCLC targets. Then Cytoscape software (ver. 3.8.1) was used to construct drug and disease target network diagram to show the relationship between α-solanine and NSCLC.
Construction Of Ppi Network And Selection Of Core Genes
The obtained candidate targets of α-solanine and NSCLC were uploaded to online STRING11.0 (https://www.string-db.org/) to obtain forecasted protein-protein interactions (PPI), with the species set as "Homo sapiens". Then download the PPI network file and import it into Cytoscape for topology attribute analysis. CytoNCA was used to calculate degree centrality (DC), median centrality (BC), proximity centrality (CC), eigenvector centrality (EC), network centrality (NC) and local mean connectivity (LAC). Based on the results above the median of the six parameters, the core target of the network was obtained.
Gene Ontology and Pathway Enrichment Analysis for NSCLC-Related Targets of α-solanine
The candidate targets of α-solanine and NSCLC were bioinformatics analyzed by R software. Gene Ontology (GO) function enrichment and KEGG pathway analysis were performed using BiocManager, Dose, Cluster Profiler and Enrichment Plot in R software. The top 20 items with the highest degree were presented in the form of bubble diagram.
Experimental Validation
Cell Culture and Drug Preparation
Human Lung cancer A549 and PC-9 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Inc.) with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc.) and 1% streptomycin and penicillin (Gibco), and maintained at 37˚C in a humidified atmosphere containing 5% CO2. The α-solanine (J & K Scientific) were dissolved in dimethyl sulfoxide (DMSO, Sigma-aldrich). The control group was treated with DMSO only under identical conditions.
Cell Viability Assay
In this study, 3x103 lung cancer cells were inoculated into 96-well plates, and 0, 10, 12, 14, 16, 18, 20, 22, 24 µg/mL concentrations of α-solanine with the same volume of DMSO were added after 24 h; At different time points during treatment, 10 µl CCK‑8 (Dojindo) solution was added to the wells and incubated at 37˚C for 2h, and the optical density (OD) was measured by a plate reader (SpectraMax M5; Molecular Devices LLC) at the read mode of absorbance (450 nm).
Cell Migration And Invasion Assays
For cell migration assays, transwell filters (Costar) were uncoated with Matrigel on the upper surface of a polycarbonic membrane (6.5 mm diameter, 8 µm pore size). After being cultured for 24h in medium containing various concentrations of α-solanine (0, 12, 18 and 24 µg/mL), A549 and PC-9 cells (2×105) were resuspended in 200 µL of serum-free medium in the upper chambers, while 600-µL medium containing 10 % FBS (as a chemoattractant) was added to the bottom chamber. After incubating for 24 h at 37°C in a humidified incubator with 5 % CO2, cells in the upper chamber were carefully removed with a cotton swab. Cells that had migrated to the basal side of the membrane were fixed with 4% polyformaldehyde, stained with crystal violet, mounted and dried at room temperature for 30 minutes. Then randomly selected three visual fields and counted and recorded the number of cells invading the Matrigel (3.9 µg/µL, 60–80 µL) with an inverted microscope at ×100 magnification. Each test was performed in triplicate. With a similar principle and approach, we used Transwell filters with coated Matrigel on the upper surface of a polycarbonic membrane for a cell invasion assay.
Apoptosis Analysis By Flow Cytometry (Fcm)
Apoptosis was detected according to the manufacturer's instructions. Briefly, 3x105 A549 and PC-9 cells were inoculated into 6-well plates, after being cultured for 24h, then treated with 0, 12, 18, 24 µg/mL concentrations of α-solanine with the same volume of DMSO. After 24h of treatment, the cells were collected and washed with phosphate buffered saline (PBS), resuspended in binding buffer, labeled by Annexin V-FITC, incubated with propidium (PI), and apoptosis was detected by flow cytometry.
The Detection Of Targeted Energy Metabolite
After treated with α-solanine for 24 hours, A549 cells were collected and extracted with 80% methanol [25]. The supernatant was removed and analyzed by LC-MS/MS system (HPLC, Shim-pack UFLC SHIMADZU CBM30A system, MS/MS, Tandem mass spectrometry, Qtrap®6500+). Samples were separated using SeQuant ZIC-pHILIC(5 µm, 2.1×100 mm) at 40°C. Mobile phases were made up of mobile phase A (10 mmol/L ammonium acetate + 0.3% ammonia solution,) and mobile phase B (90% acetonitrile water). The elution gradient was as follows: 5:95 V/V at 0 min, 50:50 V/V at 9.5 min,5:95 V/V at 11.1 min, 5:95 V/V at 14.0 min. The flow rate was 0.4 mL min− 1 with an injection volume of 2 µL. The MS parameters were as follows: electrospray ionization temperature: 450°C; mass voltage: 5500 V (positive ion mode) or -4500 V (negative ion mode); ion source gas: GS I 40 psi, GS II 55 psi; curtain gas: 35 psi; collision-activated dissociation: medium. The mass detection was optimized based on declustering potential and collision energy.
Immunofluorescent Staining
A549 and PC-9 cells (2×105) were inoculated into 6-well plate (Corning), after being cultured for 24h in order to make the cells adhere and stabilize. Then the test group treated with α-solanine (18 µg/mL), an equal volume of DMSO was used as control. After 24h of treatment, the cells were collected and washed with PBS, then cells were fixed with 4% polyformaldehyde, and permeabilized with 0.1% Triton X-100 made in PBS for 15–20 minutes. Prepare a blocking solution of 5% normal goat serum in PBS for 30 minutes, then incubated with relevant antibodies (Dilute with 5% goat serum) overnight at 4℃. Apply an appropriate fluorophore-conjugated secondary antibody diluted in antibody dilution buffer to the coverslips and incubate for 1 hour in a moist, dark environment. Mount cover slips on microscope slides with Hydromount (National Diagnostics) containing DAPI (if desired) for nuclear staining.
Protein Extraction And Western Blot Assay
In brief, A549 and PC-9 cells were treated with 0, 12, 18 and 24 µg/mL α‑solanine (final concentration) with the same volume of DMSO for 24 h, collected, lysed with radioimmune precipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology), quantified by BCA Protein Assay Kit (Beyotime Institute of Biotechnology), and separated by 10% SDS-PAGE. The proteins were transferred onto 0.22 µm PVDF membrane (Bio-Rad). The membranes were then blocked with 5% non-fat milk for 1 hour at room temperature, then incubated with relevant antibodies overnight at 4℃ against GPI (1:1,000, Proteintech, 15171-1-AP), ALDOA (1:1,000, Proteintech, 11217-1-AP), TPI1 (1:1,000, Proteintech, 10713-1-AP), PKLR (1:1,000, Proteintech, 22456-1-AP), LDHA (1:1,000, Proteintech, 19987-1-AP), ALDH3 (1:1,000, Proteintech, 15578-1-AP) and GAPDH (1:3,000, GENE TEX, GTX100118), washed with PBST (0.1% Tween-20, Diluted with PBS). The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit IgG, 1:3000, Beyotime, A0516) and visualized by an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.). Band density were evaluated by ImageJ (National Institutes of Health).
Statistical Analysis
Statistical analysis was processed with GraphPad Prism 8 software. Data were expressed as the mean ± SD and analyzed using Student’s t-test (for comparison between two groups) or one-way ANOVA with a post hoc Tukey test (for comparison among three or more groups). Differences between groups were considered to be statistically significant if values of P < 0.05.